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Mechanisms of anaesthetic depression of neocortical arousal El-Beheiry, Hossam El-Dean Mohamed 1990

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MECHANISMS OF ANAESTHETIC DEPRESSION OF NEOCORTICAL AROUSAL by HOSSAM EL-DEAN MOHAMED EL-BEHEIRY M.B, Ch.B., Cairo U n i v e r s i t y , 1978 M.Sc. (Anaesthesia), Cairo U n i v e r s i t y , 1982 M.D. (Anaesthesia), Cairo University 1986  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FAULTY OF GRADUATE STUDIES Department of Pharmacology  & Therapeutics,  Faculty of Medicine  We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1990 © HOSSAM EL-BEHEIRY, 1990  In presenting this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of Pharmacology and Therapeutics The University of British Columbia Vancouver, Canada  Date 28 dune 1990  DE-6 (2/88)  ii  ABSTRACT  The  most  widely  accepted  i n t e r r u p t conscious  processes  or  synaptic  by  potentiating  putative  transmitters  arousal  include  primary  objective  hypotheses  inhibition, especially  i n the n e u r o l o g i c a l  here  was  to  a n a e s t h e t i c s may obtund t h i s  cellular  recording  techniques.  Bath  synaptic the  systems t h a t and  T  determine  the  neuronal  pig  generate  -aminobutyrate  neocortex  of  0.5-2.5  (in  associated  with  concentrations relevant  ( 0 . 5 - 1 . 5 MAC  Because  slices),  including  potentials  by  which  a small in  of  these  and  However, the  did  they  repetitive  intra-  preparation,  concentration  conductance  10-200 LIM)  using  anaesthetics,  hyperpolarization  input  neurons  microiontophoretic,  dissimilar  alveolar  of u n c o n s c i o u s n e s s  activities  The  (MAC)  and  mV)  which  The  lower  (3-5  (10-30%). agents  not  which  are  significantly  produced  striking  spike  firing  in  most  affect  decreases evoked  by  ( e l e c t r i c a l ) s t i m u l a t i o n or i n t r a c e l l u l a r c u r r e n t i n j e c t i o n s . the  reduction  investigated  and  membrane p r o p e r t i e s .  spontaneous  orthodromic  the  increase  to the production  the p a s s i v e in  an  (GABA).  mechanisms  vitro  structurally  minimum  10-1300 nM, r e s p e c t i v e l y , produced was  The  neocortical  were c a r r i e d out on p y r a m i d a l  pharmacological,  applications  of  excitation  neocortex.  i s o f l u r a n e - a halogenated e t h e r , and A l t h e s i n - a s t e r o i d a l concentrations  anaesthetics  arousal.  and V o f guinea and  general  in  glutamate  The m a j o r i t y o f the i n v e s t i g a t i o n s IV  that  i n t h e b r a i n by d e c r e a s i n g  acetylcholine,  in layers  suggest  observed in  neuronal  extensively (EPSPs  and  changes  excitability, on  IPSPs).  i n d u c e d a dose-dependent,  i n membrane the  excitatory The  properties  could  effects  anaesthetics  and  application  r e v e r s i b l e depression  of  inhibitory of  isoflurane  in the  not  explain were  postsynaptic or  amplitude  Althesin of  EPSPs,  i i i with E C s  o f 1 MAC and ~50 uM, r e s p e c t i v e l y .  5 Q  i n a dose-dependent EPSPs  by  the  manner.  In  GABA-activated  EPSP-IPSP  sequence,  tionally  applied.  Despite  depressed  the  electrical In  EPSPs  a  responsiveness depression,  of  to  as  the  possibility  the neuronal  applications  acetylcholine,  hyperpolarizing  IPSP  evoked  of  the  in  the  was  anaesthetics  addi-  strongly  by  subpial  be  to  somatic  the  involved  in  markedly  changes  and  the  evoked  by  applications  depressed.  of  effects  (NMDA).  GABA  were  not  observed  with  its  Seme degree  of  selectivity  f o r t h e i s o f l u r a n e - and A l t h e s i n -  induced  acetylcholine  Under were  in  vitro  of  responses  conditions  t o t a l l y blocked  of  known  increase  associated with Because  the  or d i s p o s i t i o n ,  in  2+ Ca  -activated  of  the  compared  responses  depression  in  the  t h i s may be of importance  anaesthetic  requirements  with to  glutamate.  acetylcholine  responses  t o GABA  i n t h e mechanism  in  clinical  for  syndromes  hypomagnesaemia. genesis  of  synaptic  the i n t e r a c t i o n s of  afterhyperpolarizations a  hypomagnesia  and the o r d e r  and g l u t a m a t e was r e v e r s e d ; the  to  the  dendritic  a l s o was e v i d e n t from t h e l o w e r E C ^ s depressions  of EPSP  reduced  N-methy1-D-aspartate  depolarizing  slightly  attenuation  t o a c e t y l c h o l i n e , g l u t a m a t e . and GABA  glutamate  whereas  postsynaptic  may  conductance  responses  a p p l i c a t i o n were  a  administration  associated  significantly  dendritic  substances  Anaesthetic  and  of  that  sensitivities  depolarizations  affected  the  epileptiform a c t i v i t i e s  transmitter  determined.  The  IPSP-blockade,  the  bicuculline,  A  reduced  shunting  produces  GABA -antagonist,  this  well  that  were  stimulation.  cognizance  were  as  IPSPs a l s o  order to e l i m i n a t e p o s s i b l e  Cl-conductance  observed  The  (AHPs).  + K -conductance  transients anaesthetics  is  a f f e c t e d by  were i n v e s t i g a t e d  The AHPs which a r e produced were  suppressed  Ca  by  the  2+ on  influx spike  s p e c i f i c a l l y by  anaesthetics  in  a  IV  dose-dependent manner under conditions where contaminating blocked  by  bicuculline.  Since  the  passive  membrane  IPSPs had been properties  unaffected, an interference with a transmembrane Ca -influx may +  were  be involved  in the anaesthetic actions. The  effects of anaesthetics  increases  in  hippocampal  intraneuronal  on glutamate-induced and voltage-dependent 2+ 2+ Ca ([Ca ].) were determined in cultured  neurons with a Ca-sensitive  probe  (Fura-2) and  microspectro-  fluorometric techniques. Isoflurane application depressed the increases in 2+ [Ca ]. produced by application of glutamate under conditions where its actions would be  favoured at NMDA- and  +  quisqualate-subtypes  of  receptors.  2+  K -induced  increases  in  [Ca  ]•• also  were  reduced  by  application  of  isoflurane, probably due to actions on voltage-dependent Ca-channels in the membrane. These investigations have provided excitatory transmitter anaesthesia.  A  actions  plausible  evidence for the  in neocortex are  mechanism  would  first  selectively  include  time that  depressed  suppression  of  postsynaptic Ca-conductances associated with the AHPs and glutamatergic, well as cholinergic interactions at pre- and post-synaptic involved in neocortical arousal.  by the as  sites on neurons  V  TABLE OF CONTENTS  CHAPTER  TITLE PAGE ABSTRACT  Page  1 ii  TABLE OF CONTENTS  v  LIST OF TABLES  x  LIST OF FIGURES  xi  ACKNOWLEDGEMENT  xv  LIST OF ABBREVIATIONS  xvi  DEDICATION  xvii  1 INTRODUCTION  1  1.1 G e n e r a l scope o f t o p i c  1  1.2 The c o n s c i o u s system o f t h e b r a i n 1.2.1 D e f i n i t i o n s 1.2.2 I n t e g r a t i o n o f f u n c t i o n and a c t i v i t y o f t h e c o n s c i o u s subsystems 1.2.3 F u n c t i o n a l neuroanatomy o f t h e c o n s c i o u s n e s s system 1.2.4 N e o c o r t i c a l a c t i v a t i o n 1.2.5 A c t i v a t i n g t r a n s m i t t e r s i n t h e n e o c o r t e x 1.2.6 The a c t i v a t i n g t r a n s m i t t e r s and t h e i r i o n i c mechanisms of action 1.2.6.1 A c e t y l c h o l i n e 1.2.6.2 S-Glutamate  2 2  1.3 The a n a e s t h e t i c s t a t e 1.3.1 H i s t o r i c a l i n t r o d u c t i o n and d e f i n i t i o n s 1.3.2 S i t e o f a c t i o n o f a n a e s t h e t i c s i n t h e c o n s c i o u s n e s s system 1.3.3 Mechanisms o f a c t i o n o f g e n e r a l a n a e s t h e t i c s 1.3.3.1 N o n - s p e c i f i c ( l i p i d ) hypotheses 1.3.3.2 S p e c i f i c ( p r o t e i n ) hypotheses 1.3.3.3 A n a e s t h e t i c m o l e c u l a r mechanisms and c e l l u l a r excitability 1.3.3.4 A n a e s t h e t i c a c t i o n s on c y t o p l a s m i c membrane s i t e s  3 3 6 7 8 8 11 13 13 14 16 16 20 23 25  -vi-  CHAPTER  Page  1.4 Hypotheses and O b j e c t i v e s 2 METHODS  27 27  2.1 A n i m a l s  27  2.2 S l i c e p r e p a r a t i o n  28  2.3 S o l u t i o n s and drugs  29  2.4  31  I n t r a c e l l u l a r r e c o r d i n g arrangement  2.5 Computer a n a l y s i s  33  2.6 I o n t o p h o r e t i c t e c h n i q u e s 2.6.1 E l e c t r o d e s 2.6.2 S o l u t i o n s and equipment 2.6.3 E x p e r i m e n t a l p r o c e d u r e s  35 35 36 37  2.7 I n t r a c e l l u l a r C a measurement 2.7.1 Neuronal c u l t u r e s 2.7.2 S t o r a g e and l o a d i n g o f F u r a - 2 AM 2.7.3 I n t r a c e l l u l a r C a measurements 2.7.4 C a l i b r a t i o n of t h e system  37 37 38 38 40  2.8 S t a t i s t i c a l a n a l y s i s  40  2 +  2 +  3 SPONTANEOUS ACTIVITY, REPETITIVE FIRING AND PASSIVE MEMBRANE PROPERTIES  40  3.1 R e s u l t s 3.1.1 E f f e c t s on spontaneous a c t i v i t y and evoked r e p e t i t i v e firing. 3.1.2 E f f e c t s on Vm and membrane e l e c t r i c a l p r o p e r t i e s  40  3.2 D i s c u s s i o n  49  4 EFFECTS ON SYNAPTIC TRANSIENTS 4.1 R e s u l t s 4.1.1 R e s t i n g membrane p r o p e r t i e s  41 43  50 50 50  -VI1-  CHAPTER  Page  4.1.2 4.1.3 4.1.4  Dose-response d e p r e s s i o n of EPSPs EPSP a t t e n u a t i o n i n t h e presence of I P S P - b l o c k a d e E f f e c t s on o r t h o d r o m i c a l l y and i n t r a c e l l u l a r ^ evoked spikes 4 . 1 . 5 Dose-response r e l a t i o n s h i p f o r IPSP s u p p r e s s i o n d u r i n g C s - b l o c k a d e of t h e K-conductances  4.2 D i s c u s s i o n 4.2.1 A n a e s t h e t i c d e p r e s s i o n of EPSPs 4.2.2 A n a e s t h e t i c a c t i o n s on t h e IPSPs 5 ANAESTHETIC INDUCED ALTERATIONS IN NEURONAL RESPONSIVENESS TO ACTIVATING TRANSMITTERS AND RELATED SUBSTANCES 5.1 R e s u l t s 5.1.1 C o n t r o l r e s p o n s e s t o i o n t o p h o r e t i c a l l y a p p l i e d agents 5.1.1.1 A c e t y l c h o l i n e 5.1.1.2 Glutamate and N - m e t h y l - D - a s p a r t a t e 5.1.1.3 Gamma-aminobutyrate and b a c l o f e n 5.1.2 A n a e s t h e t i c induced a l t e r a t i o n s i n r e s p o n s e s t o e x c i t a t o r y and i n h i b i t o r y t r a n s m i t t e r s u b s t a n c e s 5.1.2.1 Responses t o ACh, G l u and NMDA 5.1.2.2 Responses t o GABA 5.1.3 S e l e c t i v i t y i n the d e p r e s s i o n o f responses t o t r a n s m i t t e r substances 5.1.3.1 A n a e s t h e t i c i n t e r a c t i o n s w i t h t r a n s m i t t e r s 5.1.3.2 Time c o u r s e of a n a e s t h e t i c a c t i o n s 5.1.3.3 D o s e - r e s p o s n e r e l a t i o n s h i p s 5.1.3.4 A n a e s t h e t i c e f f e c t s on the p o t e n t i a t i o n of G l u - r e s p o n s e s by ACh 5.2 D i s c u s s i o n 5.2.1 Responses t o t r a n s m i t t e r and r e l a t e d agents 5.2.1.1 A c e t y l c h o l i n e 5 . 2 . 1 . 2 G l u t a m a t e and N - m e t h y l - D - a s p a r t a t e 5.2.1.3 Y - a m i n o b u t y r a t e 5.2.2 A n a e s t h e t i c induced a l t e r a t i o n s i n the c h e m o s e n s i t i v i t y of e x t r a c e l l u l a r l y a p p l i e d t r a n s m i t t e r agents 6 EFFECTS OF HYPOMAGNESIA ON TRANSMITTER AND ANAESTHETIC ACTIONS 6.1 R e s u l t s 6.1.1 E f f e c t s o f bath a p p l i c a t i o n of M g - f r e e ACSF  51 54 58 58 61 65 68  70 70 70 70 72 74 81 81 87 87 87 93 93 97 97 97 101 102 102 104 109 109 110  -viii-  CHAPTER  Page  6.1.2 6.1.3 6.1.4 6.1.5  E f f e c t s of g r a d u a l removal Of [ M g ] E f f e c t s of i n c r e a s i n g [ M g ] E f f e c t s of external C a and Mg e x c l u s i o n on the G l u r e s p o n s e s A n a e s t h e t i c a c t i o n s d u r i n g Mg-free p e r f u s i o n  110 114  2 +  0  2 +  2 +  2+  119 119  6.2 D i s c u s s i o n 6.2.1 S u p p r e s s i o n of t h e ACh-induced responses 6.2.2 Changes i n [ M g ] do not s i g n i f i c a n t l y a f f e c t Glu-induced responses 6.2.3 G A B A - d e p o l a r i z a t i o n s are c o n s i s t e n t l y a t t e n u a t e d by t h e removal o f [ M g ] 6.2.4 Enhancement o f neuronal e x c i t a b i l i t y by t h e removal of [ M g ] 6.2.5 A n a e s t h e t i c - i n d u c e d d e p r e s s i o n o f G l u and GABA r e s p o n s e s i n M g - f r e e media  123 123  2 +  0  2 +  0  2 +  0  2 +  7 ANAESTHETIC-INDUCED ATTENUATION OF POSTSPIKE AHP MEDIATED BY Ca-ACTIVATED K-CONDUCTANCE 7.1 R e s u l t s 7.1.1 D e p r e s s i o n o f AHPs 7.1.2 B i c u c u l l i n e - b l o c k a d e of i n h i b i t o r y p o s t s y n a p t i c  potentials  7.2 D i s c u s s i o n 7.2.1 Mechanism(s) of a n a e s t h e t i c i n t e r f e r e n c e w i t h AHP 7.2.2 S i g n i f i c a n c e o f AHP i n h i b i t i o n i n n e o c o r t i c a l neurons generation 8 EFFECTS OF ISOFLURANE ON THE GLUTAMATE- AND POTASSIUM-INDUCED INCREASES IN INTRANEURONAL CALCIUM CONCENTRATION 8.1  Results 8.1.1 Responses t o ACh and c a r b a c h o l 8.1.2 E f f e c t s o f i s o f l u r a n e on r e s t i n g [Ca^l^ and G l u actions 8.1.3 I s o f l u r a n e a c t i o n s on K-evoked i n c r e a s e s i n [ C a ] j 2 +  8.2 D i s c u s s i o n 9 GENERAL DISCUSSION  124 125 126 127  127 127 128 133 133 137 137  138 138 139 139 144 144 147  CHAPTER  Page  9.1 N e o c o r t i c a l u n i t a c t i v i t y and g e n e r a l a n a e s t h e s i a  148  9.2 A n a e s t h e t i c a c t i o n s : d e p r e s s i o n o f e x c i t a t i o n o r p o t e n t i a t i o n of i n h i b i t i o n ?  148  9.3 The a n a e s t h e t i c s t a t e and s u p p r e s s i o n mechanisms  149  9.4 I o n i c mechanisms  of  consciousness  of a n a e s t h e t i c a c t i o n  9.5 M o l e c u l a r mechanisms o f a n a e s t h e s i a — controversy  152 the l i p i d / p r o t e i n 155  10 SUMMARY AND CONCLUSIONS  156  11 REFERENCES  160  -X-  LIST OF TABLES  TABLE  1  Page  A n a e s t h e t i c d e p r e s s i o n of neuronal r e s p o n s e s t o t r a n s m i t t e r substances  91  2  E f f e c t s o f M g - f r e e media on s e n s o r i m o t o r neurons  113  3  E f f e c t s o f i s o f l u r a n e on [ C a 2 ] - i n c r e a s e s evoked by g l u t a m a t e and K  143  +  1  +  -xiLIST OF FIGURES  FIGURE  1  Page  A n a e s t h e t i c d e l i v e r y system and r e c o r d i n g chamber used i n these i n v e s t i g a t i o n s .  30  Diagram showing t h e n e o c o r t i c a l s l i c e and s i t e s o f r e c o r d i n g and s t i m u l a t i o n and t y p e s of i o n t o p h o r e t i c e l e c t r o d e s u s e d .  32  3  O r g a n i z a t i o n of e x p e r i m e n t a l s e t - u p f o r data a c q u i s i t i o n and r e t r i e v a l .  34  4  I s o f l u r a n e and A l t h e s i n induced d e p r e s s i o n of spike a c t i v i t i e s .  2  5  Anaesthetic-induced increases genesis.  display, spontaneous  in the t h r e s h o l d f o r  42 spike 44  6  Anaesthetic interference with the r e p e t i t i v e f i r i n g a b i l i t i e s o f n e o c o r t i c a l neurons.  46  7  A l t e r a t i o n s i n t h e p a s s i v e membrane p r o p e r t i e s by a p p l i c a t i o n of i s o f l u r a n e and A l t h e s i n .  47  E f f e c t s of i s o f l u r a n e on t h e i n p u t r e s i s t a n c e of an a n t e r i o r c i n g u l a t e neuron.  48  9  EPSP-depression  52  10  Dose-dependent by i s o f l u r a n e .  8  11 12  13  induced by i s o f l u r a n e and A l t h e s i n suppression  o f the a m p l i t u d e and r a t e o f decay 53  A l t h e s i n - i n d u c e d dose-dependent d e p r e s s i o n o f t h e a m p l i t u d e s and r a t e s of decay of n e o c o r t i c a l evoked EPSPs.  55  E p i l e p t o g e n i c a c t i v i t y and EPSP s u p r e s s i o n a p p l i c a t i o n s i n t h e p r e s e n c e o f GABA-ergic n e o c o r t i c a l neurons.  56  by a n a e s t h e t i c blockade i n  Anaesthetic i n t e r f e r e n c e with the s y n a p t i c a l l y - i n d u c e d spikes and d i r e c t l y - e v o k e d a c t i o n p o t e n t i a l s by i n t r a c e l l u l a r c u r r e n t pulse i n j e c t i o n s .  59  -xii -  FIGURE  Page  14  Internal  C s - a p p l i c a t i o n i n n e o c o r t i c a l neurons.  60  15  I P S P - a t t e n u a t i o n by i s o f l u r a n e and A l t h e s i n a f t e r K conductance b l o c k a d e by i n t r a c e l l u l a r a p p l i c a t i o n o f C s .  62  Dose-dependent i n t e r f e r e n c e w i t h the IPSPs i n n e o c o r t i c a l neurons w i t h c o n c o m i t a n t b l o c k a d e of K - c o n d u c t a n c e s by intracellular Cs .  63  +  +  +  16  +  +  17  E f f e c t s of i o n t o p h o r e t i c a l l y a p p l i e d a c e t y l c h o l i n e on n e o c o r t i c a l neurons.  71  18  Glutamate evoked r e s p o n s e s i n n e o c o r t i c a l neurons.  73  19 20  NMDA-induced d e p o l a r i z a t i o n s i n s e n s o r i m o t o r l a y e r V neurons. A h y p e r p o l a r i z i n g response t o GABA a p p l i e d from a compound e l e c t r o d e assembly.  75 76  D i f f e r e n t t y p e s o f d e p o l a r i z i n g responses evoked by d e n d r i t i c a p p l i c a t i o n o f GABA.  77  C h a r a c t e r i s t i c s o f t h e GABA-evoked d e p o l a r i z a t i o n s i n n e o c o r t i c a l neurons.  79  B i c u c u l l i n e , i n h i g h d o s e s , b l o c k e d t h e GABA-induced d e p o l a r i z a t i o n s i n n e o c o r t i c a l neurons.  80  24  Depression  by i s o f l u r a n e of responses t o a c e t y l c h o l i n e .  82  25  Depression  by A l t h e s i n o f r e s p o n s e s t o a c e t y l c h o l i n e .  83  26  Depression  by i s o f l u r a n e of responses t o g l u t a m a t e .  84  27  A n a e s t h e t i c d e p r e s s i o n o f r e s p o n s e s t o g l u t a m a t e and NMDA.  85  28  P o t e n t i a t i o n o f g l u t a m a t e a c t i o n s and a t t e n u a t i o n of a c e t y l c h o l i n e r e s p o n s e s by i s o f l u r a n e .  86  E f f e c t s o f i s o f l u r a n e a p p l i c a t i o n on e q u i - a m p l i t u d e r e s p o n s e s t o GABA, g l u t a m a t e and a c e t y l c h o l i n e .  88  E f f e c t s of A l t h e s i n a p p l i c a t i o n on t h e a c e t y l c h o l i n e - , GABAand g l u t a m a t e - e v o k e d r e s p o n s e s .  89  D e p r e s s i o n o f a c e t y l c h o l i n e , glutamate and GABA r e s p o n s e s by h i g h dose of i s o f l u r a n e .  90  21 22 23  29  30  31  -xii* i -  FIGURE  32 33  34  35 36 37 38  Page  S e l e c t i v e a n a e s t h e t i c depression of responses.  acetylcholine-evoked 92  Time c o u r s e s f o r a n a e s t h e t i c d e p r e s s i o n o f j u s t - m a x i m a l r e s p o n s e s t o GABA, glutamate and a c e t y l c h o l i n e i n t h e presence o f TTX.  94  P o o l e d data show dose-response r e l a t i o n s h i p s f o r i s o f l u r a n e i n d u c e d d e p r e s s i o n s of d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r substances.  95  Dose-response r e l a t i o n s h i p s f o r A l t h e s i n - i n d u c e d d e p r e s s i o n d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r s u b s t a n c e s .  of 96  S e l e c t i v i t y i n the i s o f l u r a n e - and A l t h e s i n - e v o k e d d e p r e s s i o n of neuronal r e s p o n s i v e n e s s t o a c e t y l c h o l i n e , g l u t a m a t e and GABA.  98  C o n t i n u o u s r e c o r d of i s o f l u r a n e - i n d u c e d d e p r e s s i o n a c e t y l c h o l i n e - p o t e n t i a t i o n of g l u t a m a t e - a c t i o n s .  99  of t h e  A l t h e s i n - i n d u c e d d e p r e s s i o n of the a c e t y l c h o l i n e - p o t e n t i a t i o n of g l u t a m a t e - a c t i o n s .  100  39  B l o c k a d e of a c e t y l c h o l i n e and GABA a c t i o n s i n M g - f r e e  perfusate.  Ill  40  P o t e n t i a t i o n o f NMDA responses i n t h e M g - f r e e c o n t a i n i n g TTX.  solution  41  by g r a d u a l  bathing  112  Depression [Mg2 ] .  of g l u t a m a t e - r e s p o n s e s  removal  of  42  Depression  o f GABA-evoked d e p o l a r i z a t i o n by low [ M g 2 ] .  43  Gradual p o t e n t i a t i o n of a c e t y l c h o l i n e - a c t i o n s due t o a s e q u e n t i a l i n c r e a s e of [ M g ] .  +  0  +  0  0  GABA a c t i o n s were g r a d u a l l y p o t e n t i a t e d by s e q u e n t i a l in [Mg2 ] .  increase  +  0  45  116 117  2 +  44  115  118  D o s e - r e s p o n s e c u r v e s showing t h e e f f e c t s of [ M g ] on t h e d e p o l a r i z a t i o n s evoked by a c e t y l c h o l i n e , g l u t a m a t e and GABA.  120  46  E f f e c t s o f changing actions.  121  47  A n a e s t h e t i c a c t i o n s on a c e t y l c h o l i n e , g l u t a m a t e - and GABAinduced responses during a p p l i c a t i o n of Mg-free media.  2 +  Q  C a - and M g - c o n c e n t r a t i o n s  on g l u t a m a t e  122  -xiv-  FIGURE  48 49 50  51  52  53  54 55 56  Page  I s o f l u r a n e induced an a t t e n u a t i o n of t h e a f t e r h y p e r polarizations. Depression Althesin.  129  of the a f t e r h y p e r p o l a r i z a t i o n s by a p p l i c a t i o n s  of 131  Dose-response r e l a t i o n s h i p s f o r the d e p r e s s i o n o f the a f t e r hype r p o l a r i z a t i o n s produced by a p p l i c a t i o n s o f i s o f l u r a n e and Althesin.  132  I s o f l u r a n e and A l t h e s i n induced d e p r e s s i o n s o f t h e a f t e r h y p e r p o l a r i z a t i o n s where b i c u c u l l i n e m e t h i o d i d e had been a d d i t i o n a l l y a p p l i e d .  134  Changes i n r e s t i n g membrane p o t e n t i a l , i n p u t r e s i s t a n c e , a f t e r h y p e r p o l a r i z a t i o n a m p l i t u d e o r d u r a t i o n induced by i s o f l u r a n e and A l t h e s i n .  and 135  P o t e n t i a t i o n of g l u t a m a t e - i n d u c e d i n c r e a s e s i n i n c u l t u r e d hippocampal neurons by 0.5 MAC i s o f l u r a n e under conditions favouring Quis-receptor-subtype stimulation.  140  I s o f l u r a n e (1.75 MAC) i n h i b i t i o n of i n c r e a s e s i n [ C a ] - j produced by g l u t a m a t e a p p l i c a t i o n under NMDA c o n d i t i o n s .  141  I s o f l u r a n e (2.5 MAC) b l o c k a d e o f i n c r e a s e i n [ C a ] j g l u t a m a t e a p p l i c a t i o n under Quis c o n d i t i o n s .  142  2 +  2 +  by  V o l t a g e - g a t e d i n c r e a s e i n [ C a ] j produced by K bolus i n j e c t i o n s were depressed by i s o f l u r a n e a d m i n i s t r a t i o n under Quis conditions. 2 +  +  145  ACKNOWLEDGEMENTS I  wish  to  thank  all  the  members  of  the  T h e r a p e u t i c s who o f f e r e d h e l p and a d v i c e . g r a t i t u d e t o Dr. K. L e i g h t o n f o r h i s MacLeod  and Dr.  Baimbridge  for  B.  Sastry  providing  for  help  the  of  Pharmacology 3  I p a r t i c u l a r l y wish t o e x p r e s s my  support.  their  me w i t h  Department  I am a l s o  grateful  and encouragement  facilities  to  and  measure  t o Dr.  B.  to  K.  Dr.  intraneuronal  calcium concentration. I  would l i k e  analysis Ms.  t o thank  Mr.  of p a r t of t h e d a t a .  J . Swetnam and Ms.  L. I  Corey also  M. Wong f o r  for  his  assistance  in  the  computer  a p p r e c i a t e t h e e f f o r t and p a t i e n c e their  help  in  typing  and  printing  of the  f i n a l d r a f t of t h e t h e s i s . Last  but not  least,  I  wish  to express  would l i k e t o thank him f o r a l l h i s  my g r a t i t u d e t o Dr.  E.  Puil.  h e l p , a d v i c e , guidance and s u p p o r t .  c o n t r i b u t i o n s are g r a t e f u l l y acknowledged.  I His  -xvi-  LIST OF ABBREVIATIONS Acetylcholine Afterdepolarization Afterhyperpolarization 2-ami no-5-phosp h o n o v a l e r a t e 4-aminopyridine Y-aminobutyrate Ampere( s) A r t i f i c i a l cerebrospinal f l u i d C y c l i c guanosine monophosphate 2,4-dinitrophenol Effective concentration E f f e c t i v e dose Excitatory postsynaptic potential E x t r a c e l l u l a r calcium concentration E x t r a c e l l u l a r magnesium c o n c e n t r a t i o n Glutamate Inhibitory postsynaptic potential Input r e s i s t a n c e I n t r a c e l l u l a r free calcium concentration I n t r a c e l l u l a r f r e e magnesium c o n c e n t r a t i o n Kainate N-methy 1 - D - a s p a r t a t e Minimum a l v e o l a r c o n c e n t r a t i o n Minute(s) Ohm(s) P r o t e i n kinase C Quisqualate R e s t i n g membrane p o t e n t i a l R e t i c u l a r a c t i v a t i n g system Second(s) Standard d e v i a t i o n S t a n d a r d e r r o r o f mean T e t r a e t hyl ammoni um Tetrodotoxin Volts  -XVI1-  In t h e name of God, Most G r a c i o u s , Most M e r c i f u l "Say:  He i s God, the One and Only.  He b e g e t t e t h n o t , nor i s He b e g o t t e n .  God the E t e r n a l , A b s o l u t e .  And t h e r e i s none L i k e unto H i m . " ( T r a n s l a t i o n from the Holy  I w i s h t o d e d i c a t e t h i s t h e s i s t o my p a r e n t s , N a b i l a and Mostafa  El-Beheiry,  who have s t r u g g l e d c o n t i n u o u s l y f o r my w e l l - b e i n g .  Qur'an)  1  H.  INTRODUCTION G e n e r a l scope o f t o p i c  1.1  During  the  anaesthetics However,  past  on  two decades,  vertebrate  there  are  no  by which t h e s e  two  reasons  main  thoroughly (CNS).  studied  for  this.  on  neocortical  neurons  Certain technical  more  readily  c u l t u r e d neurons. the  chemical  steroids  and  Comprehensive dissimilar  inorganic reports  cell  have  in  the  sites  a modest  for  that  system  indicates  of  the  or  tissue  of a common s p e c i f i c i t y i n  possess  the  same p h y s i o l o g i c a l  been  anaesthetic  structures  barbiturates,  the  are  investigations  ethers,  compared  not  understanding  subcortical  an absence  There  evidence  have l i m i t e d t h e p r e v i o u s  hydrocarbons,  on t h e  state.  neuronal  nervous  target  have  the  central  the  of  described.  have  are  neurons  general  actions  (metabolic)  likely  of  been  explain  biochemical  compounds  anaesthetics  anaesthetic types  there is  that  neurons  actions  produce t h e u n c o n s c i o u s  of a r o u s a l  of  of  adequately  d i f f i c u l t i e s and  Secondly,  hundreds  and  studied  structure  that  First,  relevant  p h y s i o l o g i c a l mechanisms the  invertebrate  agents  Neurophysiological  agents.  to  and  virtually  hypotheses  mechanisms  that  1  EL-BEHEIRY  pyrimidines,  anaesthetic  actions  of  type  neuron  of  property.  structurally are  rare,  d e s p i t e a p e r c e i v e d i m p o r t a n c e of such e x p e r i m e n t a t i o n . In t h e thetics,  present  r e s e a r c h , the e f f e c t s of  isoflurane --  two c h e m i c a l l y d i s t i n c t  anaes-  a r e l a t i v e l y recent, v o l a t i l e agent,  and A l t h e s i n  --  a s t e r o i d a l p r e p a r a t i o n , were s t u d i e d on n e o c o r t i c a l neurons  of t h e b r a i n  in  order t o address (a)  do  f o u r fundamental  structurally  questions:  different  e f f e c t s on the same neuronal  anaesthetics type?  have  similar  (depressant)  H. EL-BEHEIRY (b)  i s a n a e s t h e t i c - i n d u c e d d e p r e s s i o n o f neuronal  a c t i v i t y in  due t o an a t t e n u a t i o n of e x c i t a t i o n o r an enhancement o f  2  neocortex prevailing  i n h i b i t i o n a t synapses ( o r b o t h ) ? (c)  by  what  neuronal  mechanisms  do  anaesthetics  obtund  neocortical  arousal? (d)  what  are  the  plausible  ionic  mechanisms  for  the  anaesthetic  i n t e r f e r e n c e with e x c i t a t o r y or i n h i b i t o r y processes? A major i.e.,  hypothesis  general  anaesthetics  activities  in  processes.  Neuronal  cortical  fully  the  during  the  course  act with varying  neocortex  by  of  degrees  selectively  of  the  the b a s i s  how c o n s c i o u s n e s s  investigations,  o f e f f i c a c y on  depressing  external  and  certain  internal  obtunded, r e s u l t i n g i n t h e u n c o n s c i o u s  understand  these  neuronal  electrical  e x c i t a b i l i t i e s a r e d e c r e a s e d i n a s p e c i f i c manner and a  awareness  consequently  evolved  is  for  brought  this, about  a synopsis in  environments  state.  of  In o r d e r t o more  current  unanaesthetized  are  evidence  brain  is  as  to  presented  below. 1.2  The C o n s c i o u s System In The B r a i n 1.2.1  is  Definitions.  probably  consciousness CNS;  this  is  the is  Among  most  all  difficult  phenomenological  somewhat  aspects  analogous  to and  of  human b e h a v i o u r  define. is  This  regarded  to the property  of  as  is  partly  an  attempts  have met w i t h some i n the processes  to  visualize  success  the  modular  (Kissin  1986).  producing consciousness  i n f u n c t i o n and e l e c t r i c a l of  consciousness  activity.  organization,  as  because  a t t r i b u t e of  "luminescence"  c o n s i d e r e d an a t t r i b u t e o f c u l t u r e d luminous b a c t e r i a (White Recent  consciousness  which  is  1987).  a multicomponent  The v a r i o u s  the  subsystems  o f t h e b r a i n are c l o s e l y  system involved  integrated  As a consequence o f t h e i n t e r d e p e n d e n c y  consciousness  is  usually  experienced  as  a  H. EL-BEHE IRY single, tion,  though  integrated  consciousness  components. specific  entity.  has  Awareness  objective  in  turn  in discrimination  "activation"  and  the m u l t i c o m p a r t m e n t a l  (alertness)  may  be  and  vague,  o f energy"  In  t h a t has  q u a l i t a t i v e l y , as  conceptualiza-  subjective  (awareness)  general  content  or  a d d i t i o n , consciousness  is  i.e.,  (self-awareness).  c h a r a c t e r i z e d by a " l e v e l or  In  3  in  been r e f e r r e d t o as  "affect".  Thus  a beam o f  "arousal" awareness  may be f o c u s e d i n one d i r e c t i o n o r another under t h e paradigm o f " a t t e n t i o n " . These  components  o f awareness The  above  which  —  activation, alertness,  and a t t e n t i o n - -  terms  describe  constitutes  a  represent d i f f e r e n t aspects  different  separate  and  dimensions  discrete  d e f i n e d a n a t o m i c a l and p h y s i o l o g i c a l 1.2.2  Integration  subsystems.  integrated  arousal  brought  associated,  on  activation,  in  about an  by  the  almost  motivationally  is  basis  in  For  with  and an  of  control  the  example,  an  of  of a  conscious  increase  excitation is  comparable  internal  and  alertness  external  of d r i v e - o r i e n t e d behavioural  in  activation  a t t e n t i o n onto t h e c o g n i t i v e l y  the  in  Therefore,  electrophysiological into  are  usually  increases  attention.  epiphenomenally  f o c u s e d through  sequence  the  each  subsystems o f c o n s c i o u s n e s s  in motivational  produces  events  under  facets  consciousness.  consciousness,  activity  activity.  translated  significant  such t h a t t h e f i n a l  and  awareness  is  Awareness  and  one-to-one  arousal  which  process  the v a r i o u s  increase  alertness,  brain,  awareness.  an  of  of  dual  system i n the b r a i n .  function  function  motivational-emotional of  of  As mentioned above,  closely  a r o u s a l , . a f f e c t , the  and and  enviroments  r e s p o n s e s can be  produced o r r e p e a t e d . 1.2.3 Activating reticular  Functional systems activating  of  neuroanatomy the  brain.  pathways  that  of These  the are  consciousness generated  by  p r o j e c t from the m e d i a l  system the  (a)  ascending  portion  of  the  H. EL-BEHEIRY  brainstem  into  portions  the  dorsal  o f the b a s a l  neocortex  (Moruzzi  reticular  activating  brain. cortex  Hence, can  a  of  activity  the  activity)  or  and  the  system  high  M o r i son  controls  impulses  and  the  background  for  the  the  locus  coeruleus  (very  y-  1948;  diffuse  is  whereby  l i m b i c system and most  (RAS)  of  RAS  locus  thalamus  Magoun  maintained  'pacemaker' in  ganglia,  state  be  medial  is  radiate  to  all  significantly,  to  the  Dempsey  'energy  state  coeruleus  of  r e f l e c t e d by  of  The  in  the  (Webb  the  the  1962).  levels'  activation  conscious  4  cerebral  1983).  pons.  the  The  Electrical  presence  of  6 - (high  a c t i v i t y ) waves i n t h e c o r t i c a l l y m o n i t o r e d  EEG  (Snyder and S c o t t 1972). (b)  Alertness  consciousness relaxed  subsystem.  when  state  the  (Kissin  RAS  is  1986).  Alertness mildly  from  the  behavioural  a c t i v a t i o n of t h e awareness (c)  Awareness  self-awareness activating  thalamus, nucleus  level  of of  the  hypothalamus,  the g l o b u s p a l l i d u s  basalis  of Meynert  of  the  brain.  as  behavioural  in  the  a-waves awareness  but  which  The g e n e r a l  the dorsal  are  generally  general  connected system  i n an  consciousness i n a syndrome external  integrated  in the substantia  fashion  to  provide  and a t t e n t i o n a l a c t i v i t i e s . known  stimuli  as  (Girvin  a k i n e t i c mutism, 1975).  The  to  the basal  ganglia  innominata of the  all  the  in this  i n which  there  self-awareness  and  no  system  is  the  the the  disperse,  system can is  and  paleocortex.  p r i m i t i v e elements  Lesions  the  consists  median and a n t e r i o r n u c l e i o f  and the putamen of  is  below).  The i n d i v i d u a l a n a t o m i c a l u n i t s o f t h i s system, though a n a t o m i c a l l y operate  This  reflects  of  awareness  of  resting,  i n t h e EEG.  consist  Both  level  so-called  may be c o n s c i o u s  These  brainstem.  systems and t o each o t h e r .  of the p o s t e r i o r  induces  the  system by the RAS o f t h e b r a i n (see  systems  subsystems  active  An i n d i v i d u a l  u n a t t e n t i v e d u r i n g which t h e thalamus different  is  of  result  reaction  to  responsible  H. EL-BEHEIRY  f o r t h e awareness of  the  brain  of t h e  that  i n t e r n a l enviroment  are  involved  in  a c c o r d i n g t o Mesulam and Geschwind the d i e n c e p h a l o n , lobes.  substantia  The l a t t e r  paleocortical  the  i.e.,  o f t e n behaves  at  a  patient  as though  may  shave  p a t i e n t may d r e s s o n l y h a l f of h i s  complex, b r a i n s t e m g e n e r a l substantia  innominata  produces  generalized  would  be  attention  The  to  these  self-concept effectively The  (3)  system  sing  and  unless  it  were  of events assesses  ties and  (cf.  is  of  his  Geschwind  not  face  of  the  or  exist  another  thalamo-basal  and  focused can  lobe  Such  critical  events  the  mobilizes  events  all  directly  reserves  to  in  follows:  that of  into deal  system  activation  events  be r e c o n s t r u c t e d as  environmental  septi,  self-awareness  awareness. on  gangliar  accumbens  d i r e c t s more s p e c i f i c a s p e c t s  together  (4)  the  Kissin  l o b e r e c e i v e primary contiguous  alertness  (2)  and  are  the The  deemed  consciousness the  activated  w i t h them  most  .  neocortex  mechanisms  of  (1)  and  the world d i d  side  inferior  state  significant,  events,  one  of  system and t h e n u c l e u s  posterior  sequence  apparatus  biologically  inferior parietal  in patients with u n i l a t e r a l  one-half  only  of  t o e i t h e r a s t r o k e o r tumor.  Activation  awareness  and  meaningless  environment.  septi  body.  The a t t e n t i o n a p p a r a t u s .  a  accumbens  i n n o m i n a t a and t h e p o s t e r i o r  parts  self-awareness  Heilman and Watson (1977) and Mesulam  The i n d i v i d u a l  (d)  The  r e c e i v e c h o l i n e r g i c p r o j e c t i o n s from t h e d i e n c e p h a l i c  centres.  i.e.,  of  (1978) are t h e n u c l e u s  i n t h e i n f e r i o r p a r i e t a l l o b e secondary  all,  self.  phenomenology  (1978) have d e s c r i b e d a c h a r a c t e r i s t i c b e h a v i o u r lesions  "one's  5  site  1986). sensory  hypothalamus motivational  of  these  complex  consciousness-directing  The sensory c o r t e x and t h e (processed)  as w e l l  information  as t h e basal  ganglia,  emotional/evaluation,  from  i n f e r i o r temporal the  thalamus  for cognitive  respectively.  and  proces-  Integrated  H. EL-BEHEIRY  i n f o r m a t i o n from t h e s e c o n t r o l c e n t r e s f l o w s t o the p r e f r o n t a l t u r n c o n t r o l s the f l o w t h r o u g h  the amygdaloid-hippocampal  the a t t e n t i o n - d i r e c t i n g a c t i v i t i e s . thalamic  feedback  unattended  arrangement  area which  complex,  i.e.,  in for  In a d d i t i o n , t h e r e e x i s t s a p r e f r o n t a l -  which  sensory c h a n n e l s .  6  inhibits  electrical  Thus a t t e n t i o n can  activity  be c o n c e n t r a t e d  in  the  towards  a  system  is  given sensory m o d a l i t y . 1.2.4  Neocortical  characterized  by  et a l .  which  1988)  a  activation.  steady is  state  A  functioning  level  prerequisite  of to  consciousness  cortical  the  excitation  fast,  'phasic'  i n f o r m a t i o n b i d i r e c t i o n a l l y i n t h e a t t e n t i o n system. results  from s u b c o r t i c a l  excitatory 1989). layers  transmission  (Pauly  et al  1989)  neurons  in  activation  may  1986). be  originating the b a s a l  forebrain  ( L e v i t t and Moore  (Rothballer  biogenic  to  the  ascending  1978;  sleep-waking  Lidov cycle  e t a l . 1980) (Aston-Jones  has  coexistence  for  the  Rustioni  1967;  1956;  Ling  Divac  as i n d i c a t e d by  (Reader  1981;  in neocortical  and  neurons  and  Foulks  1959;  demonstrations  in  the  e t a l . 1988).  midbrain Adren-  systems t o t h e n e o c o r t e x have been d e s c r i b e d  More  interpreted  and Lewis  influences  amine-containing  maintenance o f c o n s c i o u s n e s s . been  and  vertical)  Lehmann e t a l . 1980; Kimura e t a l .  and pons t h a t p r o j e c t d i f f u s e l y t o t h e c o r t e x e r g i c and s e r o t o n e r g i c  Giuffrida  (Shute  R e i n e r and McGeer 1987) of  or  from t h e m i d b r a i n , pons, thalamus  and h i s t a m i n e r g i c  significant  s p e c i a l i z e d groups  activation  (horizontal  1981a;  of  r e t i c u l a r pathways p r o j e c t t o t h e deep c o r t i c a l  Adrenergic  Wanatabe e t a l . 1984;  Oshima  transfer  Neocortical  intracortical  and  Hoover and J a c o b o w i t z 1979;  Spencer e t a l  of  as  (Enzure  C h o l i n e r g i c ascending  magnocellular 1975;  as w e l l  (Matsumura  w i t h a c e t y l c h o l i n e i n RAS neurons  although and  these  Bloom  a r e more  1981)  than  r e c e n t l y , d i r e c t morphological of  the  neuropeptide  ( V i n c e n t e t a l . 1983).  related to  the  evidence  substance  P  H. EL-BEHEIRY 1.2.5 (ACh)  Activating  is  considered  neocortical  of  a  major  a c t i v a t i o n can  a t r o p i n e (Funderburk has  neurotransmitters  electrical  and Case 1951).  stimulation  prolongs  and  prolonged  induce  of  of  duration  repetitive  firing  Another (Krnjevic contrast  likely  major  and Phi 11 i s to  ACh,  Glu  information  1987). the  at  the  rate  of  transfer  and  Rustioni  1989).  individual. unconscious  of  blocked  to  synapses  responsiveness  firing  evoked  glutamatergic  Also, be  For  in  McLennan rapid  are  by  pathways)  more  numerous  exogenous ACh  prerequisite  in  the  neocortex  1983;  Puil  onset/offset  pathways  for  is  can  to  glutamate  and Benjamin  action.  f o r the f a s t t r a n s f e r  Glu,  as w e l l  the  conscious  example,  in  (Conti a  et  comatose  al.  (Glu)  1988). acting  In as  a  as  processing  processing  (Krnjevic  i n t r a c o r t i c a l l y , as  structures  Hence, the p r o c e s s i n g  well  1987;  patient,  modulates as  between  Giuffrida the  than t h a t p r e s e n t  and  tonic i n an  ACh alert  o f sensory i n f o r m a t i o n i s obtunded i n the  state.  be e l u c i d a t e d .  circuitry  by  ( K r n j e v i c e t a l . 1971a).  information,  subcortical  A c o n t r i b u t i o n of  (Enzure  neuronal  1988).  e x c i t a t i o n may be a t t e n u a t e d t o a l o w e r l e v e l  to  because  However, a s c e n d i n g c h o l i n e r g i c e x c i t a t i o n of t h e n e o c o r t e x  cortical  yet  and  enhanced  bursts  believed  a  transmitter'  spike  which  is  of  (likely  1987,  1963a;  synapses  the  inputs  such t h a t  t r a n s m i t t e r , may be r e s p o n s i b l e of  a state  enhance  transmitter  has  Acetylcholine  with cholinomimetics  (Krnjevic  g e n e r a t i o n of mnemonic t r a c e s  neocortex.  'activating  of  synaptic  afterdischarges in  ACh  the  A r o l e f o r ACh a t n e o c o r t i c a l  generation  Applications  and  cortical  be e l i c i t e d  been suggested i n t h e neurons.  in  7  inhibitory However,  transmission some  reports  t h a t may make up t h e elementary  and Oshima  1981b).  In  addition  to n e o c o r t i c a l suggest  structure to  activation  a model  of  has  neuronal  of  cortical  activation  excitatory  neurons,  inhibitory  H. EL-BEHEIRY GABAergic  neurons  identified 1978a,  have  during  b;  postulated  stimulation  Enzure  important  been  and  transmitter  of  Oshima  the  in  RAS  in  1981a).  responsible  Acetylcholine  transmitters  mechanisms: of  awake  for  a  negative  these  cats  have  been  (Inubushi  GABA  etal.  could  feed-back  be  loop  an  in  the  system.  The ' a c t i v a t i n g t r a n s m i t t e r s ' and t h e i r i o n i c mechanisms o f a c t i o n 1.2.6.1  other  model;  Consequently,  neuronal o r g a n i z a t i o n o f t h e c o n s c i o u s n e s s 1.2.6  the  8  (a)  is  a decrease  repolarization  conductance,  brought  (c)  due  (ACh). about  in  to.  Excitation  through  the  resting  reduction  of  K  following  by  the  delayed  ACh  ionic  conductance,  +  in  (b) K  or  by  membrane a  slowing  rectifying  +  a r e d u c t i o n i n the slow a f t e r h y p e r p o l a r i z a t i o n (AHP) due t o +2  inhibition  the  mediated  the  slow  Ca  + -activated  K  conductance  and  (d)  inhibition  of t h e M - c u r r e n t . (a) ates  Decrease  the  resting  K  conductance.  +  K r n j e v i c " and  his  associ-  (1971a) f i r s t demonstrated t h i s mechanism i n n e o c o r t i c a l neurons  lightly  anaesthetized c a t i n in vivo experiments.  by Woody  et  consciously strated  al.  (1978)  freely  the  cingulate input  in  moving  actions  c o r t e x of  resistance  potential  observed.  Since in  reduction  in  potential  close  produced  large  cats.  of  ACh  guinea  to  reversible  rectification  confirmed  In  increase  during  close  -100  was  these  neurons,  or to  neurons  pigs.  to  K  +  -100  positive  no  and in  an mV  convincing the  mV).  results  the  the  slices  of  ions  of be  of  ACh  also of  ACh  anterior  best have  injections  equilibrium  and  for  of  a  were  (anomalous)  explained an  the  also  actions  inward  in  demon-  a tendency  depolarizing  could  (both  CI"  (1986)  studies  evidence  Intracellular in  Prince  application for  investigations  actions  in v i t r o  the e a r l y  conductance  shifts  unconventional  McCormick  on  there  CI"  these  Subsequent  of t h e  by  a  equilibrium CI"  potential  which and  the  H. EL-BE HE IRY  IPSP  reversal  neuronal that  potential  responses  ACh  to  probably  did  not  ACh.  acted  obviously  Therefore, by  change  Krnjevic  reducing  the  character  et a l .  membrane  9  (1971a)  conductance  of  the  concluded  for  K .  As  +  2+ supporting  evidence,  w i t h movements cortical (b) 1971a;  of K  Ba  , which  interfere  i n a v a r i e t y of e x c i t a b l e t i s s u e s ,  +  Reduction  in  Woody e t a l .  "slowing  the  delayed  1978;  down"  of  K*  Bernardo  application  rather  specifically  was shown t o e x c i t e  of  Ba^  Depression  of  rectifier.  (Krnjevic  +  i.e.,  afterdepolarization observations  the  was  Ca  activated  1971b)  falling  K  phase  This  conductance.  t h e AHP.  substantially  (ADP).  (Krnjevic  et a l .  the  showed t h a t when ACh was a p p l i e d by p r e s s u r e neurons  effect  (Krnjevic  mimicked leads  of  the  by  to  a  action  r e p e t i t i v e f i r i n g and a f t e r d i s c h a r g e s . 2+ +  net outward c u r r e n t a b o l i s h e s  neocortical  This  and P r i n c e 1 9 8 2 ) , w h i c h i s  repolarization,  p o t e n t i a l s , and enhances  of t h i s  known t o  c e l l s i n a manner s i m i l a r t o ACh ( K r n j e v i c e t a l . 1971b).  iontophoretic  (c)  is  McCormick  ejection,  reduced  finding  is  and  in  e t a l . 1971a, 1978a,b).  The  depression  and P r i n c e  the postspike even  agreement  However,  replaced with  (1986) AHP  in  by  an  the  the exact  early  mechanism  o f the AHP d e p r e s s i o n i s not w e l l u n d e r s t o o d ; 2+ by an i n c r e a s e d Ca binding to calmodulin 2+  t h e e f f e c t c o u l d be mediated 2+ (a Ca - b i n d i n g p r o t e i n ) , a  reduction  Ca-sequestration  i n Ca  -influx,  be due t o a more the  relevant  replacement depolarizing  direct  diminished interaction  K-ionophores of  the  event  AHP  (cf.  by  mediated  an  internal  between t h e  Krnjevic ADP  by  may 2+ Ca  obscured o r b l o c k e d by t h e AHP (McCormick  or  it  could  a c t i v a t e d ACh-receptor  1984).  be  or  On  the  an  unmasking  due +  to  Na  which  other  ordinarily  and P r i n c e 1 9 8 6 ) .  hand, of  a  would  and the slow be  H. EL-BEHEIRY  (d)  S u p p r e s s i o n of t h e M - c u r r e n t .  t h a t the m u s c a r i n i c time-dependent,  of  tends  this  and  to  slow  charges  As  K-current  a consequence  hyperpolarize  outward  is  now good reason  lead to s e l e c t i v e suppression  non-inactivating  depolarizations. neuron  actions  There  current  and  of  the  generated  greatly  to  relatively  e x c i t a t i o n of  this  firing  and  small  current,  subsides.  f a c i l i t a t e s the  believe  of a v o l t a g e -  by  repetitive firing  10  the  Suppression and  afterdis-  i n d u c e d by c o n c o m i t a n t a c t i o n s o f e x c i t a t o r y t r a n s m i t t e r s l i k e G l u .  The M - c u r r e n t was f i r s t d e s c r i b e d i n b u l l f r o g s y m p a t h e t i c  ganglia  Adams  1982;  Malenka  olfactory  cortex  et a l .  1980),  1986;  (Constanti  then  Moore  and  Sim  in  et a l . 1987)  Gclhwiler and D r e i f u s s  hippocampus 1988), and  (Halliwell  followed  human  by  and  Adams  guinea  neocortex  pig  (Halliwell  (Brown  1986).  Because  (1982) r e p o r t e d t h a t ACh was i n e f f e c t i v e i n the  absence  2+ of  Ca  in  result  of  hippocampal a  (1977).  In  neurons,  at  least as  more  voltage-clamp  investigations  currents  and  is  recent et a l .  (1985)  ineffective in  the  apparent  properties  of  Ca-channels  is  the  v o l t a g e - and  absence  a t t r a c t i v e because  muscarinic actions discussed Cholinergic  brainstem, spinal 1974a; 1977;  Libet  The  idea  the c e r e b r a l  cortex,  ACh  Bernardo  be  Krnjevic  sympathetic  .  Hence,  the  and  membrane  primarily  explanation  on  for a l l  neurons. for  all  neostriatum,  and  a  o n l y inward-Ca 2+  acts  a unitary  reported  Zieglgansberger  Dodd e t a l . 1981;  rat  reflect  levels  Reiter  and P r i n c e  1982;  of the  thalamus,  c o r d and s y m p a t h e t i c and p a r a s y m p a t h e t i c  1970;  by  e x t r a c e l l u l a r Ca  that  i t provides  been  of  may  are r e a l l y C a - a c t i v a t e d K - c h a n n e l s  t h u s f a r f o r CNS  i n h i b i t i o n has  including  of  earlier  blocks  time-dependencies  Ca-channels.  M-current  speculated  found t h a t muscarine  they c o n c l u d e d t h a t the " M - c h a n n e l s "  system,  the  K-conductance  Belluzzi  their  of  Ca-dependent  ganglia,  that  part  1974;  nervous  hypothalamus,  ganglia  (Krnjevic*  Hartzell  et a l .  ffrench-Mullen  et a l .  1983).  Inhibition  response,  has  been  i n many c a s e s  .  observed  just  Cholinergic  w i t h i n the s u p e r f i c i a l layers  (II  prior  to  H. EL-BEHEIRY  11  the  ACh  depolarizing  i n h i b i t i o n had been observed  and I I I )  of t h e c e r e b r a l c o r t e x  mostly  (Krnjevic  1974a).  I t i s a l s o p r e s e n t i n l a y e r V, a l t h o u g h the i n h i b i t o r y r e s p o n s e s  neurons  in  the  superficial  layer  are  considerably  larger  (McCormick  in and  P r i n c e 1986).  In the n e o c o r t e x , c h o l i n e r g i c i n h i b i t i o n may be a consequence  of  interneurons  GABA-ergic  which  impinge  on  the  impaled  McCormick and P r i n c e The slowness  (cGMP)  i n ACh  does  not  neurons  (Krnjevic  actions  of  ACh  by  neuron  the  thereby  extracellularly  applied  producing  (Haas  a c t i o n on c o r t i c a l system  1982;  mimick  et a l .  neurons  suggests  an  involvement  i n t h e t r a n s d u c t i o n of t h e r e s p o n s e .  the  muscarinic  1976;  Bernardo  c o u l d be mediated  actions and  of  Prince  i n t r a c e l l u l a r ^ by  dependent p r o t e i n k i n a s e i n n e o c o r t i c a l neurons 1.2.6.2  IPSPs  ACh,  1986).  o f a secondary messenger GMP  activated  ACh  in  1982). cGMP  Cyclic  subcortical However,  and by  (Woody and Gruen  a  the  cGMP-  1988).  S-Glutamate  (Glu).  The e x c i t a n t e f f e c t o f G l u i s a r e s u l t  o f t h e d e p o l a r i z a t i o n of t h e  neuron  to the t h r e s h o l d  spike  activation.  associated  An  with the  conductance change of  spike  involvement  receptor-coupled  measured  genesis.  The  during net  voltage-dependent  and  activated  respective  by t h e  N-methyl-D-aspartate and k a i n a t e  (ka).  such  another  ionophore  glutamate  change  interactions  complex  a  Glu  at  combination of  process  contributes  e x c i t a t i o n a f t e r the  behaviour of  voltage-dependent  voltage-dependent  represents  voltage-independent  the  presumed  ionic  to  The d i f f e r e n t i a t i o n between NMDA and  of  as  o f b l o c k a d e a t NMDA r e c e p t o r s by  2-amino-5-phosphonovalerate  (2-APV)  both  channels  receptors  non-NMDA  or  the  blockade  (NMDA) and a t non-NMDA r e c e p t o r s f o r q u i s q u a l a t e  has been made m o s t l y on the b a s i s antagonists  of  for  for  (Quis)  responses specific recently  H. EL-BEHEIRY  MK-801 and by Coan  Mg  et a l .  (Hwa  2 +  1987;  and A v o l i  Mayer  C o l l i n g r i d g e and L e s t e r  and  1984;  Nowak e t  Westbrook  1987;  al.  Pull  at  blockade enters  NMDA r e c e p t o r s  by  Mg  2+  is  (Nowak  a direct  et a l .  the a c t i v a t e d c h a n n e l s  thereby et a l .  conferring 1982;  a  Thomson 1986).  potentials  neurons  and  1986;  Benjamin  1988;  this  repetitively  1984).  state  a c t i v a t e d by G l u - i n t e r -  consequence This  and decreases  bistable  and a b i s t a b l e s t a t e change neocortical  Thomson  1989).  The v o l t a g e - s e n s i t i v i t y o f t h e c a t i o n i c c h a n n e l s actions  1984;  12  of  of  a  blockade  occurs  i n magnitude  the  membrane  NMDA i n d u c e s  an  lead  and  to  to  a  potential  inwardly  greater  develop  when  Mg  ?+  on d e p o l a r i z a t i o n , (MacDonald  rectifying  in the input/output behaviour can  voltage-dependent  current  of t h e neuron;  tendency  pacemaker-like  to  fire  in  action  oscillations  in  membrane p o t e n t i a l s ( F l a t m a n e t a l . 1986). The d e p o l a r i z a t i o n s potentials  near  hyperpolarized suggests t h a t + and  K .  zero with  The  the  responses  (cf.  progressive  reversal  t o changes  by NMDA and  millivolts.  both t y p e s  sensitive NMDA  evoked  in is  P a d j e n and S m i t h  The  reversal  removal  of  of agonists  potential external markedly  1981;  Puil  non-NMDA a g o n i s t s  [Na ] +  potentials  membrane  non-NMDA whereas  more  Na ;  this  +  p e r m e a b i l i t y to  responses the  reversal  become  extracellular  increase  of  have  is  reversal  affected  by  changing  the  1981).  In  cultured  spinal  much  Na  more  potential  external cord  +  [Ca  for ]  neurons,  a p p l i c a t i o n s o f Glu t h a t a c t i v a t e r e c e p t o r s o f t h e NMDA-subtype i n c r e a s e t h e 2+ 2 intracellular Ca - a c t i v i t y measured with Ca - s e n s i t i v e fluorescent 2+ probes. H e r e , Mg can be used t o d i s t i n g u i s h the NMDA-receptor-coupled 2+ 2+ Ca - i n f l u x from t h e v o l t a g e - d e p e n d e n t source o f Ca - i n f l u x t h a t also +  is Glu  i n i t i a t e d by t h e application  NMDA-actions  produces  (MacDermott  tetrodotoxin  et a l .  1986).  (TTX)-insensitive  In  neocortex,  decreases  in  H. EL-BEHEIRY extracellular  [Na ] ,  ion-sensitive agonists  —  [Ca  microelectrodes  NMDA, Q u i s  on e x t e r n a l  [K ]  and  permeable  to  due  to  extrusion  active  and  [Mg  (Pumain  ]  et a l .  ka - - a l s o  measured 1988).  produce  with  Glu  responses  K  and  +  P a d j e n and S m i t h 1983;  that by  the  a  efflux  pumping  of  K  may  +  process  the are  specific dependent  be  partly and  passive  Sonnhof  Pumain e t a l .  that or  1983;  1987).  The A n a e s t h e t i c S t a t e 1.3.1  Historical  considered  an  introduction  American  and  contribution  definitions.  since  the  Anaesthesia  first  successful  d e m o n s t r a t i o n by W.T.G. Morton took p l a c e i n M a s s a c h u s s e t t s in  selective  i n c l u d e those  (Biihrle  Pumain and Heinemann 1985;  and that  i n d i c a t i n g t h a t the a c t i v a t e d c h a n n e l s  +  are  1.3  ]  13  1846  realize  (Bigelow that  and  However,  innovation  spontaneously. Morton,  1846).  For  of  named  Aether  anaesthetic  by  properties  in  first  word  century  AD by  diethylether in  the  Its  ( c f . Smith was  Greek  has  first  examined t h e could  which  Switzerland  animals  "anaesthesia"  who  significance  Frobenius.  a l c h e m i s t named P a r a c e l s u s The  such  example,  had been d i s c o v e r e d  anyone  was  around  used  as  philosopher,  in  its  Holmes,  Oliver  c o n s i d e r a b l y e a r l i e r i n 1751 sensation" In state  (Beecher  the c l i n i c a l  this  Hospital  history hardly  agent  arisen used  Valerius  and a  will  by  Cordus  particularly physician  and  Wendell  modern  sense  in  the  who d e s c r i b e d  the  usage i n the E n g l i s h  in B a i l e y ' s English  though  Dictionary  language  it  was  defined  as  " a d e f e c t of  1968). c o n t e x t , Rees and Gray  c h a r a c t e r i z e d by  renamed  by  Dioscorides,  is  to  by  public  1986).  Its  ascribed  the  1540  described  n a r c o t i c e f f e c t s of the p l a n t Mandragora. usually  have  pharmacological  were  General  is  t r i a d e as  narcosis,  analgesia  narcosis,  reflex  (1950) and  defined anaesthesia  relaxation.  suppression  and  Gray  in  relaxation.  as  a  1960 More  H. EL-BEHEIRY  r e c e n t l y , White  (1987) attempted t o d e f i n e g e n e r a l  w h i c h the CNS i s lost  and t h a t  r e v e r s i b l y depressed  on  anaesthesia.  recovery,  Atkinson  operational)  nothing  and  definition  is  recalled relating (1987)  suggesting  surgery.  inhibition central part  of  Accordingly, of  the  synaptic the  afferent  reflex  arc.  of  the  the  in  anaesthesia  is  a  system,  depression  definition,  w i t h two main advantages.  anaesthesia  blockade  of  though  uncommon,  First,  i t avoids  of  the  the  efferent  provides the  the  different provides  an o b j e c t i v e d e f i n i t i o n a p p l i c a b l e t o humans as w e l l as e x p e r i m e n t a l  animals.  Site  Himwich  (1951)  within  the  increasing the  of was  the  brain depths  of  first  that  anaesthetics to  were  diencephalon,  and  pons  are  vertical as  recent  and  three  highly  sensory system,  nonspecific modulating located  of  sensory all  sensorimotor  essentially  within  concept  as  neurophysiology Livingstone  the  sensory  depressed  given  midline  deepens, the  vertical  the  deepest  depressed. support  for  This  interrelating  nonspecific  structures,  a CNS  systems - - t h e  and the motor system. of  by  before  (1954) v i s u a l i z e d t h e  responsibility  interrelations. the  is  Finally  have  levels,  sequentially  anaesthesia  inhibited.  system.  horizontal  and  cortex  i n t e r r e l a t e d , though  has  of  c e n t r e s o f the m e d u l l a are  the n o n s p e c i f i c  system  consciousness  reversibly  then  gradually  in  the  The c e r e b r a l  s t r a t i f i c a t i o n o f the b r a i n .  composed  specific  developments  in  the  affected  a n a e s t h e s i a o c c u r s when the v i t a l More  suggest  of a n a e s t h e s i a .  subcortical  mesencephalon  action  Secondly,  as  it  1.3.2  (or " n a r c o s i s " ) .  of  provided  reflex  This  (i.e.,  to s t i m u l i of  and  of  process  triade  coordination  is  period  the  of  i n d e f i n i n g unconsciousness  to  a mechanistic  r e f l e x response  considered  part  mechanisms  anaesthesiologist problems  they  provided  that  m o d i f i c a t i o n of the normal p h y s i o l o g i c a l by  as a s t a t e  t o such a degree t h a t c o n s c i o u s n e s s  associates  by  anaesthesia  14  extending  system from  The and is the  H. EL-BEHEIRY  m e d u l l a t o t h e d i e n c e p h a l o n , and c o n t a i n i n g t h e a s c e n d i n g  RAS.  reticular  component  control  f o r m a t i o n has of  anaesthesia  been demonstrated  consciousness,  several  be a v i t a l  investigators  have  lines  sciousness,  of  as  King  the  for  to  the  relate  (French et a l .  1953;  1956).  experimental  reflected in  Because  attempted  t o a l t e r a t i o n s i n the f u n c t i o n of the RAS  A r d u i n i and A r d u i n i 1954; Two  to  15  evidence  a  have  desynchronized  altered  this  EEG,  possible  is  concept. in  Conchronic  p r e p a r a t i o n s of e x p e r i m e n t a l a n i m a l s w i t h o u t the m i d b r a i n r e t i c u l a r f o r m a t i o n (Genovesi  etal.  anaesthetics, in  the  1969;  of  the  (1971)  firing  glucose  depressed  in  From  or  et a l . the  the  The e x a c t reasons neocortex  in  (Winters  such  in  in  reticular  (Savaki  least  Mori as  t o be  Shimoji  halothane  produced  et a l .  resulting  Moreover,  s p e c i f i c a l l y and rats  1983;  1967,  1972).  neurons.  of  some  changes  et a l .  the r e t i c u l a r c o r e  brainstem  discussion,  it  is  brainstem depression  glucose that  not  neurons  anaesthetics  found  at  a t i v i t y , b e f o r e t h e r e are  1972;  level  with  local  consistently  anaesthetized  Sakabe  in  et a l .  with 1985;  1986).  produce u n c o n s c i o u s n e s s  suggest  and  noted  e t a l . 1971,  mesencephalic  halothane  above  s i m p l y proposes  cerebral  that  u t i l i z a t i o n was  neocortex  pentobarbital Peschanski  of  be  formation  Mori  observed  can  cortical  e f f e c t s a t the u n i t a r y  increased  cerebral  changes  reticular  D a r b i n j a n et a l . 1971;  excitatory  the  Also,  i n the p a t t e r n o f  activity  and B i c k f o r d  an  1956).  neocortex  the  r e q u i r e s comprehensive  as t h e s o l e  that  such  is  as  well  the  as  likely  s p e c i f i c i t y are  neurological investigation.  any  hypothesis  s i t e a t which  needs c a r e f u l assessment.  consumption  for  apparent  In  site  of  action  of  not e x a c t l y known.  mechanisms  of  anaesthetics  summary, d a t a on  neurophysiological  the  which  local  observations anaesthetics. The r o l e  anaesthetic  of  state  H. EL-BEHEIRY  1.3.3  Mechanisms  investigators action  would  of  debate  of a n a e s t h e t i c s 1974;  cellular  and m o l e c u l a r  Peschanski  1980;  e t a l . 1984;  levels  Franks  about  and  the  physiochemical such  as  prolongation  (Krnjevic  1972). 1.3.3.1  anaesthetics structure  the  (1899).  in  Their  membrane  inhibitory  plasma the  (lipid)  classical lipids  observations  view as  were  no  is  viewed the  the  properties  i.e.,  specific that  controversy  These the  site  Overton  excellent  have  specific  postsynaptic  of  level,  release  or  a  membrane  imply  that  macromolecular or  receptor  anaesthetics  by  Haydon  neurons  highly  transmitter  on  the  e x c i t a b i l i t y by a  to  hypotheses.  the p h y s i c a l  membrane,  membrane  actions  at  A t the m o l e c u l a r  neuronal  of  1972;  1983;  on  few  site  sites  al.  molecules  of  a 1971,  target  properties  reduction  as  et a l .  et  1986).  of  Though  considerable  Dluzewski  depression  in  Non-specific  Here,  dissolution  of  a c t by c h a n g i n g  of  required.  change  Mori  of  anaesthetic  general  neocortex  exact  a matter  Bangham and H i l l  presynaptic  selective  the  1982;  of  the  1972;  1986),  Lieb  anaesthetics.  of  Mori  remain  effects  ranged w i d e l y from a very  mechanisms  general  importance  et a l .  M i l l e r 1985;  suggestions  uniform  the  of  (Robson 1967;  Krnjevic  (Richards  action  16  act  (1901)  correlation  is  through  and  Meyer  between  the  potency o f a v a r i e t y o f a n a e s t h e t i c s and t h e i r l i p i d s o l u b i l i t i e s ( o l i v e / o i l partition coefficient).  However, t h i s  c o r r e l a t i o n does not e x p l a i n  how t h e  drugs produce a n a e s t h e s i a . The e a r l i e s t a t t e m p t t o overcome t h i s d i f f i c u l t y was t h e c r i t i c a l hypothesis critical  of M u l l i n s volume  membranes.  (1954) which p o s t u l a t e d t h a t a n a e s t h e s i a  f r a c t i o n of  Mullins  anaesthetic  suggested  that  the  molecules excess  is  volume  o c c u r s when a  achieved would  volume  in  cause  neuronal lateral  p r e s s u r e on the i o n i c p o r e s o f t h e membrane w h i c h tends t o o c c l u d e them.  As  H. EL-BE HE IRY  a  result,  impulse  conduction  and  synaptic  transmission  would  A f t e r f u r t h e r m o d i f i c a t i o n s by Paton and Pertwee (1972), Seeman  and Roth  critical  (1972),  volume  Haydon  hypothesis  et a l .  provided  (1977)  and  a reasonable  Its  weakness  (e.g., same may  soaps)  way. have  ether  was the should  quite  different  an  anaesthetic,  convulsant  (Richards  assumption  be a n a e s t h e t i c s  C e r t a i n compounds  is  compounds  implicit  model  and t h a t they  w h i c h are  effects;  very  for  whereas  1980).  i n the CNS a l s o  that a l l  example,  et a l . for  considered, relations thesis In  become since  w h i c h cannot  (Halsey  in  1973).  substances  work  chemical  in  should  structure  hexafluoroisopropylmethyl  hexafluorodiethylether  be n e a r l y  compounded  i d e n t i c a l as  these  when  groups  is  both  barbiturates  show  be p r e d i c t e d from the  the  a  powerful  simple  processes  are  solubility. and  pronounced  these  The  steroids  are  structure/activity  critical  volume  hypo-  1974).  view o f t h e s e d i f f i c u l t i e s ,  by which a n a e s t h e t i c s cause  of  the  The p a t t e r n of uptake and d i s t r i b u t i o n of  further  both  explaining  all  determined o n l y by t h e p a r t i t i o n c o e f f i c i e n t and t h e l i p i d difficulties  the  soluble  should  1975),  (1978)  ( M i l l e r et a l .  lipid  similar  impaired.  Seeman (1972,  Halsey  phenomenon o f r e v e r s a l o f a n a e s t h e s i a a t h i g h p r e s s u r e s  be  17  indirect  changes  a t t e n t i o n was d i r e c t e d towards  could disrupt in  the  the  activity  structure of  of  the  functional  lipid  membrane  mechanisms bilayer  to  proteins.  Three models have been p r o p o s e d : (a)  The  phase-transition  hypothesis  suggests  that  lipids  immediately  s u r r o u n d i n g an e x c i t a b l e membrane channel a r e e x c l u s i v e l y i n a gel  phase,  w h i c h tends t o m a i n t a i n the patency  become  of  the c h a n n e l .  The  lipids  f l u i d on a d d i t i o n o f an a n a e s t h e t i c ( i . e . , t h e r e i s a p h a s e - t r a n s i t i o n ) , which  allows  the  channel  to  close  (Lee  1976).  This  hypothesis  is  H. EL-BEHEIRY  s u p p o r t e d by t h e which (Lee  finding that anaesthetics  phase-transition 1976;  Hill  development  of  occurs  1978).  In  the  in p u r i f i e d phospholipid  a d d i t i o n , high  anaesthesia  decrease  (pressure  pressure  reversal),  temperature model  the  at  membranes  antagonizes and  18  both t h e  anaesthetic-  induced d e c r e a s e i n p h a s e - t r a n s i t i o n t e m p e r a t u r e (Kamaya e t a l . 1979). (b)  The l a t e r a l  phase s e p a r a t i o n h y p o t h e s i s p o s t u l a t e s t h a t under normal  c o n d i t i o n s membrane p h o s p h o l i p i d s (Trudell to  1977).  expand  required  or if  membrane  less  without  and  expenditure  energy  than  or  gel.  An  volume t h a t r e s u l t s  when  purely  fluid  phase  forms  change.  Anaesthetics  may  "melt"  would  be  analogy  is  ice melts.  r e q u i r e d t o d e c r e a s e the volume than t o compress a  gel  of  were  p r o v i d e d by t h e r e d u c t i o n i n energy i s  i n both f l u i d  C o n v e r s i o n o f one form t o a n o t h e r p e r m i t s t h e membrane  contract with the  co-exist  the  Less  i c e o r water  gel  phase  and  t h e r e b y i n c r e a s e t h e energy r e q u i r e d t o d i s p l a c e p o r t i o n s o f the membrane and  decrease  prevent ions  the  across  the  membrane  opening the  of  lateral  compressibility.  p r o t e i n channels  postsynaptic  membrane  that or  two  distrupt lateral-phase types  of  phospholipids  separations (Trudell  p a r t i a l l y r e v e r s e d by h i g h p r e s s u r e s c)  The f l u i d i z e d - 1 i p i d h y p o t h e s i s  lateral  and  rotational  phase-transition  hypothesis,  thought to f a c i l i t a t e the  channel  motions  proteins.  this  Indeed,  i n model  et a l .  translocation  general  membranes  1975).  changes  These  may of in  anaesthe-  composed  of  effects  are  increase  the  ( T r u d e l l e t a l . 1975).  states of  decrease  conformational  proteins responsible for transmitter release. tics  permits  This  that anaesthetics  membrane  increase  in  components.  As  in  t h e membrane  disorder  the is  i o n i c - c h a n n e l c l o s u r e by r e d u c i n g t h e s t a b i l i t y o f The  enhancement  of  the  decay  of  the  miniature  postsynaptic  current  by  inhalational  f l u i d i z e d - 1 i p i d or p h a s e - t r a n s i t i o n The hypotheses fluidized  lipid  fluidity.  These  agents  is  hypothesis  H. EL-BEHEIRY  19  consistent  the  (Gage and H a m i l l  r e l a t e d t o phase t r a n s i t i o n , l a t e r a l suggest suffer  that from  anaesthetics  some s e r i o u s  with  act  phase  by  1975).  separation  increasing  constraints  and  and  membrane  inconsistencies,  such as the f o l l o w i n g : (1)  fluidity  anaesthetic  changes  O l d f i e l d 1979; an  lipid  concentrations  used c l i n i c a l l y  (2)  in  increase  which  (Boggs e t a l .  in  However,  an i n c r e a s e  on  because  in  body  other  hand,  be  detected  well  in  and L i e b  unless  excess 1978;  it  should  oppose  models.  In  1°C)  also  of  those and  temperature  consistently  and does not produce should  offset  the  contrast,  Richards  of a nerve  and  his  d i d not r e n d e r  membrane  anaesthesia.  decreases  anaesthetic  of 9 , 1 0 - t e t r a d e c a n o l ,  to the  anaesthetic  member  in  solubility,  show c o n v u l s a n t  above-  associates it  less  (1978)  susceptible  which have i d e n t i c a l fluidity  One isomer l o w e r s and the o t h e r e l e v a t e s  phase t r a n s i t i o n t e m p e r a t u r e of a p h o s p h o l i p i d some c h e m i c a l congeners  action  agents;  isomers  ( P r i n g l e and M i l l e r 1978).  anaes-  anaesthesia;  a n a e s t h e t i c p o t e n c i e s do not induce s i m i l a r changes i n membrane  (5)  the  Turner  increases  the f l u i d i z i n g e f f e c t a c c o r d i n g  t o b l o c k a d e by a n a e s t h e t i c t h e c i s and t r a n s  than  and t h e r e f o r e s h o u l d enhance  cooling  demonstrated t h a t c o o l i n g  (4)  are  Franks  (less  (Eger e t a l 1965)  the  mentioned  used  1976;  temperature  ( H a r r i s and Groh 1985)  (3)  are  cannot  L i e b e t a l . 1982);  fluidity  t h e t i c potency  bilayers  or isomers,  chemical  and  activity.  membrane;  d i f f e r i n g only t r i v i a l l y  physical  the  properties  Examples  of  such  molecular  from an as  lipid  modifica-  H. EL-BEHEIRY tions  that  convert  an  anaesthetic  to  a  convulsant  20  molecule  include  a d d i t i o n of a methyl group to the a l i p h a t i c side chain of b a r b i t u r a t e s , addition of a single double bond in the s t e r o i d nucleus of  alphaxalone  and replacement of one f l u o r i n e atom i n halothane by a hydrogen  atom  ( c f . L a b e l l a 1981, 1982). 1.3.3.2  Specific  (protein)  hypotheses.  Because  the  actions  of  anaesthetics on membrane l i p i d s do not appear to explain t h e i r p h y s i o l o g i c a l effects,  it  is  natural  to consider  the p o s s i b i l i t y  that anaesthetics  d i s r u p t membrane function by d i r e c t i n t e r a c t i o n with the proteins membrane.  may  of  the  Anaesthetic molecules may combine with a hydrophobic region of a  protein molecule, induce a conformational change  and thereby make i t  less  active  hypothesis  that  (Eyring  et a l .  1973).  A  more  complex  suggests  d i f f e r e n t sets of hydrophobic s i t e s e x i s t on target p r o t e i n molecules, with each of the hydrophobic s i t e s accomodating anaesthetic molecules of d i f f e r e n t sizes  (Richards  etal.  1978).  General  anaesthetics  have  been  shown  to  induce changes in conformation and p r o p e r t i e s for a wide v a r i e t y of p u r i f i e d proteins and protein structures such as actomyosin (Strosberg microtubular protein of an axopod ( A l l i s o n et a l . 1970), genase  (Hulands  etal.  Featherstone 1967),  1975),  myoglobin  l u c i f e r a s e (Franks  and  glutamic  haemoglobin  and Lieb 1984;  et a l .  1972),  dehydro-  (Schoenborn  1985),  and  albumin and  l a c t o g l o b u l i n (Balasubramanian and Wetlaufer 1966), calcium-dependent ATPase ( P r i c e et a l . 1974),  and receptors f o r a c e t y l c h o l i n e (Young  Aronstam et a l . 1986), dopamine, ouabain, or opioids as well (Baker  as a-adrenergic and  anaesthetic  ligands  Schapira  1980).  molecules  and  (Fairhurst The  functional  (Labella et a l .  and L i s t o n 1979)  kinetics proteins  of  interaction often  et a l .  1978; 1979),  and aequorine between  the  i n d i c a t e competitive  antagonism according to L a b e l l a ' s (1981) observations on anaesthetic-induced  H. EL-BEHEIRY  perturbations opioid,  of  specific  digitalis  supported  by  the  purified  cardiac  Ca  the  of  1974;  and  dopamine  non-competitive  of  myofibrillar  et a l . and  brain  a  (Labella  and  force  inhibition  of  et a l .  ligands  for  1979).  This  competitive  isolated  there  of  induced-depression  the  of  However,  mixed  membranes  halothane  ATPase  isometric 1975).  to  receptors  observations  maximal  Price  binding  21  are  enzyme  of  muscle  reports  activity  is the  antagonism  cardiac  other  the  by  by  (Price  indicating  anaesthetics.  F o r example, d i e t h y l e t h e r , m e t h o x y f l u r a n e , t r i c h l o r o e t h y l e n e , c h l o r o f o r m and halothane  show " m i x e d "  k i n e t i c s i n i n h i b i t i n g b r a i n and e r y t h r o c y t e  cholinesterase a c t i v i t y (Braswell results there  of are  Hydrophobic  other  structural,  clearly  functional  distinguishable  with high  tional  change,  marked  s t r u c t u r a l changes  although  pockets  at  higher  e.g.,  occur  and L i e b 1984)  between  the  regions above  6-lactoglobulin;  proteins  (or  of chemical clefts  1964).  of  two,  where  one  Wishina  and  Pinder  Whether  may  suggest  sites  on  or  et a l .  around  more 1966).  can  1979).  a  such  binding  Other  substrate  or  to b i n d .  sizes),  There  perhaps  anaesthetics The  flexibility  may  hydrophobic  bovine  reflects  serum a  bind  many s i t e s of low a f f i n i t y has y e t t o be e s t a b l i s h e d .  also  (e.g.  surfaces  albumin;  single  are  intermediate  bind in a nonhyperbolic p a t t e r n are l e s s (e.g.,  cause  e t a l . 1977 and l u c i f e r a s e ;  structures  or  and  no c o n f o r m a -  t o be c o n s i d e r a b l y more  certain  proteins.  anaesthetics  fold  that  the p r o t e i n s  little  (Harkey  Sachsenheimer  and have p o o r l y d e f i n e d s t o i c h i o m e t r y Pinder  studies  binding  hemoglobin  and t h e r e appears  grooves where a n a e s t h e t i c s  and  of  concentrations,  in  where  which a l l o w s a w i d e r range hydrophobic  types  binding  s e l e c t i v i t y o r cause  c o f a c t o r ( e . g . , adenylate kinase; Franks  and  This discordance with the  p o c k e t s can r e s u l t from i n e f f i c i e n t f o l d i n g o f  can b i n d a n a e s t h e t i c s  hydrophobic  and K i t z 1977).  acetyl-  large  or  discrete Wishina patch  or  H. EL-BEHEIRY  The well  consequences  understood.  of  anaesthetic  Nonetheless,  binding  three  to  basic  protein  function  mechanisms  have  22  are  less  been  proposed  target  protein  ( c f . M i l l e r 1985): (a) c o m p e t i t i o n f o r c o f a c t o r s needed f o r a c t i v a t i o n , (b)  minor  sterically  induced  rearrangements  r e s u l t i n g from b i n d i n g t o hydrophobic (c)  allosteric effects  require  anaesthetics  induced  to  bind  by to  in  the  s i t e s near the a c t i v e c e n t e r and,  anaesthetic different  protein  interaction  conformations  of  a  that  protein  w i t h d i f f e r e n t a f f i n i t i e s , a l t h o u g h t h i s has not been e s t a b l i s h e d . Two main c r i t i c i s m s have been launched a g a i n s t hypothesis" specific Second,  (Ueda and Kamaya  anaesthetic there  hypothesis t h a t most  is  can  account  transmission;  strating  the  macromolecules. oil/water  the l i p i d  the  however,  This  that  no e v i d e n c e  for  possibility  The l a r g e  suggest  s t u d i e d thus  synaptic  olive  binding  virtually  proteins  1984).  of  for  the " p r o t e i n  number of p r o t e i n s non-specificity  the mechanism  pressure  reversal  far  no r e l a t i o n t o  bear  they  binding  of  by  favoured.  which It  those  as  t h a t show  is  phenomenon.  can be c o n s i d e r e d  specific  perturbation  protein is  true  involved  models  anaesthetics  for to  illu-  protein  s i t u a t i o n i s s i m i l a r t o the use of membrane models  partition  hypotheses.  coefficients  R e c e n t l y , however,  to  demonstrate  the  a s p e c i f i c binding  of  of halothane  to  1986).  The  agreement  volume  hypothesis  non-specific precisely  by  the  theory)  predictions  of  and t h e p r e s s u r e  assuming  a  single  the  reversal  molecular  m o l e c u l e b i n d s t o , and i n a c t i v a t e s a s i n g l e a d d i t i o n , pressure increase  in  critical  data c o u l d  model site  in  which  (Franks  excitability,  which  counteracts  a  impressive  be more one  anaesthetic  causing  generalized  (a  obtained  and L i e b 1982).  r e v e r s a l c o u l d be e x p l a i n e d by p r e s s u r e  and  feasibility  b r a i n m u s c a r i n i c r e c e p t o r s has been r e p o r t e d (Aronstam between  in  In  a general  decrease  in  H. EL-BEHEIRY  e x c i t a b i l i t y caused by a n a e s t h e t i c b i n d i n g t o s p e c i f i c p r o t e i n s i t e s et a l .  1975,  1978)  t h e i r target  sites.  1.3.3.3 In  order  to  question  or  to  a  molecular  how l i p i d  few  reports  have  mechanisms and neuronal Haydon  and  being  hypothesis  for  "squeezed  anaesthetic  or p r o t e i n p e r t u r b a t i o n s  i n c e l l u l a r e x c i t a b i l i t y should answer,  anaesthetics  (Kendig  away"  from  A n a e s t h e t i c m o l e c u l a r mechanisms and c e l l u l a r e x c i t a b i l i t y .  accept  about  simply  23  be a d d r e s s e d .  attempted t o  could  Because  explore  the  actions,  produce  this  link  depression  is d i f f i c u l t  between  molecular  (1977)  reported  a  decrease  in  capacitance  t h i c k e n i n g o f b l a c k l i p i d membranes  i n d u c e d by a l k a n e s and a n a e s t h e t i c s  dose-dependent  same group  c)  using  L a t e r , the  a decrease when  an  using  currents  input  anaesthetic  the  between  in  same  applied  techniques,  anaesthetic by 50%,  was  an  the  published  increase bathing  i n membrane  solution.  a Meyer-Overton  needed  to  the m i s s i n g  membrane  bilayers  b,  was a s s o c i a t e d w i t h  block  thickness)  Urban  type  of  evoked  (1985),  correlation  inward  and t h e i r membrane/buffer p a r t i t i o n c o e f f i c i e n t s .  t h e i n h i b i t i o n of e x c i t a b i l i t y . lipid  in  concentrations  be suggested t h a t t h i s i s  the  (i.e.,  a,  i n s q u i d axon found t h a t an  s t a t e i n a c t i v a t i o n of N a - c h a n n e l s  capacitance  and in a  (Haydon and Urban 1983  two-electrode voltage-clamp techniques  i n c r e a s e i n the steady  to  depression.  associates  manner.  the  l i n k between the l i p i d  sodium It  may  hypotheses  and  F o r example, a n a e s t h e t i c m o l e c u l e s f l u i d i z e thereby  inducing  membrane  thickening  and  d e s t a b i l i z e t h e open i o n i c c h a n n e l s a c t i v a t e d d u r i n g e x c i t a t i o n . Franks lipid several  and L i e b (1978) u s i n g  bilayers  showed  anaesthetics.  no  change  X-ray in  d i f f r a c t i o n studies thickness  following  of m u l t i l a m e l l a r applications  Fernandez e t a l . (1982) found t h a t i n the s q u i d  c h l o r o f o r m reduces t h e magnitude o f t h e N a - g a t i n g  of  axon,  c u r r e n t s but has no e f f e c t  H. EL-BEHEIRY on  their  kinetics.  In  contrast  chloroform  increases  t r a n s l o c a t i o n of the l i p o p h i l i c i o n d i p i c r y l a m i n e . t h a t general  anaesthetics  of i o n i c channels  It  the  rate  More r e c e n t e v i d e n c e showed t h a t membrane c a p a c i t a n c e of  neurons  had been  input  increased  Puil  membrane  inputs  i n g can  survive  mammalian  in  the  central  depression  unlikely closing  some  induced  by  t h a t are b e l i e v e d t o be d e v o i d  of  ganglion  of  neurons  o f membrane t h i c k e n -  activated  that  will  from s q u i d  be  axon  encountered  are c o n t r a d i c t o r y  to  in  those  Haydon and Urban, 1983a, b, c ) . gap  e x c i t a b i l i t y was  specifically  difficulty  observations  the  (Carlen  r e p o r t e d an i n c r e a s e of t h e  Hence, i f t h e t h e o r y  neurons,  lipid  hippocampal  applications  the c r i t i c i s m o f e x t r a p o l a t i n g o b s e r v a t i o n s  attempt t o b r i d g e  K-current"  root  i . e . , i n neurons  (Lieberman 1976).  ethanol  (1987) a l s o  above (Haydon e t a l 1977;  Another  by  trigeminal  r e c o n c i l i n g t h e abovementioned discussed  decreased)  and Gimbarzevsky  h a l o t h a n e and i s o f l u r a n e  to  (not  capacitance  synaptic  and  charge  v i a m o d i f i c a t i o n o f the s t r u c t u r a l parameters o f the  matrix.  e t a l . 1986).  of  is therefore  a c t t o m o d i f y t h e k i n e t i c s of opening  24  between t h e m o l e c u l a r  the by  discovery  halothane  of in  a  hypotheses  novel  molluscan  and  "anaesthetic neurons  and  2+ insensitive Na  +  to 4  amino-pyridine  replacement  bind  directly  (Franks to  the  (4-AP),  and L i e b relevant  tetraethylammonium  1988). channel  that  regulates  appropriate  link  attenuation  of  including such  between  membrane  v o l a t i l e and  current  the  should  channel. the  However,  A  agents  investigated  in  and  may  not  of  abilities  where  their  and  anaesthetics it  in  the  messenger  provide  hypothesis  variety  their  cases  Co  on the second  this  large  that  stabilize  perturbation  excitability.  be  and  some p o i n t  protein  intravenous  suggested  protein  a c t i v a t e d s t a t e or a l t e r n a t i v e l y act at system  They  (TEA),  and  the the  anaesthetics to  activate  ECr 's n  are  H. EL-BEHEIRY strongly In  c o r r e l a t e d w i t h t h e i r minimum a l v e o l a r  addition,  demonstration In  this  novel  i n neurons  conclusion  anaesthetic  concentration  activated  (MAC)  values.  K-conductance  requires  of t h e CNS i n v e r t e b r a t e a n i m a l s .  the d a t a i n t h e l i t e r a t u r e does not  m o l e c u l a r mechanisms  25  of  anaesthesia  and t h e  fill  the gap  anaesthetic-induced  between  depression  of e x c i t a b i l i t y . 1.3.3.4 absence  of  a plausible  interacting 1975;  Anaesthetic  a c t i o n s on c y t o p l a s m i c membrane s i t e s .  explanation  w i t h plasma membrane  1986)  hypothesized  that  of  how a n a e s t h e t i c s  l i p i d s or p r o t e i n s , anaesthetics  could  depress  Krnjevic  act  In  the  neurons  (1972,  indirectly  by  1974b,  at  cyto-  2+ plasmic  membrane  sites.  For  example,  anaesthetics  might  depress  the  Ca  2+ accumulating  activity  of  mitochondria  and  other  cytoplasmic  Ca  -binding  2+ membranous which  proteins,  in  turn  membranes for  could  as w e l l  this  thus  increasing  influence  intraneuronal  the  conductance  as a l t e r t h e p r e s y n a p t i c  hypothesis  is  levels  of  properties  free of  and  ,  excitable  r e l e a s e of t r a n s m i t t e r s .  suggested by both b i o c h e m i c a l  Ca  Support  electrophysiologic  evidence: (a)  inhalational  1971;  Biebuyck  agents 1973;  i n h i b i t mitochondrial Rosenberg  and  activity  Haugaard  (Brunner  1973;  et a l .  Hawkins  and  2+ Biebuyck  1980).  Large  intravenous  anaesthetics  preparations  (Lee  reductions  et a l .  also  in  mitochondrial  have  1979;  been  Sweetman  observed  et a l .  p r e p a r a t i o n o f r a t b r a i n (Sweetman and E s m a i l  Ca  1981)  uptake in  and  by  in vitro in  in vivo  1975), 2+  (b)  barbiturates  neurons  of  cats  1986).  In  brain  increase in vivo  free  intracellular  (Morris  synaptosomes  and  several  Krnjevic  1985;  inhalational  h c r e aHs ea r r i ts h e1 9 8i8n) ,t r a c e l l u l a r ( [i gChae *r ^ . ;d oDs ae ns ,i e l l i nand 2  Ca  free  activity  in  CNS  Morris  et  al.  anaesthetics, 2+ Ca  in  concentration  H. EL-BEHE IRY  (c)  2,4-dinitrophenol  physiological thetics,  (DNP),  effects  on  particularly  the  1972;  K r n j e v i c e t a l . 1978a;  changes  in  i n vivo  similar  to  K r n j e v i c 1989; (e)  more  (Godfraind  or  inhibitor,  neurons  altered  and  hypoxia  metabolic  central  acids  d)  peptides  a  responsiveness  et a l . Sastry  1970;  Krnjevic  action  and  electro-  to  those  to  excitatory  1971;  preparations  of  anaesamino  Catchlove  Puil  induces  (Glotzner  K r n j e v i 6 and L e B l o n d 1989)  recently,  produces  et a l .  1978),  in vitro  anaesthetic  similar  26  excitability  1967;  LeBlond  and  and  (1988)  showed  that  halothane  in  currents  in  2+ clinically  relevant  hippocampal  CA 1 neurons  on t h e l e a k  conductances.  Although cellular that  it  basis  remain  evidence, unknown was  of  of  neuronal 1987;  anaesthetic  to  inducing  Anaesthetics  do  type  not  not  Acceptance o f t h i s  inhalational  of  a  anaesthetics,  CA1  will  cell as  well  type as  without  might  several  the  input to  an  neurons;  points  (Cohen  by  of  1973).  (or  other  experimental structures  elevated  enhanced even  Berg-Johnsen  by  [Ca *].. 2  conductances  hyperpolarization  the  depression  membranes  The  effect  critical  membrane  1974b).  any  explain  pressure  resolved  these  secondary  Miu and P u i l  hypothesis  high  been  alter  evoke  hippocampal  single  not  increase  consistently  F u j i w a r a e t a l . 1988;  by  (Krnjevic  hyperpolarization  (e.g.,  investigations  has  substantially  are  on m i t o c h o n d r i a l  molecules  mechanisms  manner  Ca  halothane-induced  reversed act  inward  hypothesis  there  example,  proteins)  biophysical  neurons  For is  elegant  actions,  how a n a e s t h e t i c s  i.e.,  suggested  this  anaesthetic  cytosolic  depressed  i n a dose-dependent  that  respiration  question  Ca-binding  seems  unanswered.  mitochondrial The  concentrations  of  the  K -efflux. +  in and  the  same  Langmoen  1989).  depend on f u r t h e r e l e c t r o p h y s i o l o g i c a l using  a  variety  a systematic  of  intravenous  biochemical  approach  and to  H. EL-BEHEIRY  elucidate  the  effects  of  the  spectrum  of  anaesthetics  on  the  27  respiratory  2+ chain  enzyme  system  combined  with  [Ca  and i n v i t r o mammalian p r e p a r a t i o n s . o u t i n nervous  tissue  ness d i s c u s s e d  above.  1.4  Hypotheses The  primary  mechanisms  However, questions 1.  of  the  by  which  this  objective  could  were  addressed:  the  the  suggesting to  anaesthetic  course  not  of  the  by  in  be  vivo  carried  of  conscious-  to  study  been  an  extensive  anaesthetics  on t h e c h e m i c a l  transmitter  substances.  was  neocortical  attained  agents  unless  the  arousal.  three  on the neuronal  basic  excita-  neurons? depression  of  neocortex  due  to  potentiated  excitation?  investigations,  anaesthetics  Accordingly,  than  study  was  of t h e a n a e s t h e t i c a major  neocortex  glutamatergic designed  to  actions?  hypothesis is  developed  more  vulnerable  stimulatory  determine  s e n s i t i v i t i e s of n e o c o r t i c a l  2 2.1  have  t h a t c h o l i n e r g i c e x c i t a t i o n i n the  depression  mechanisms  depress  What are the p l a u s i b l e i o n i c mechanisms  During  should  investigations  anaesthetics  i n h i b i t i o n o r depressed 3.  present  What are the e f f e c t s of a n a e s t h e t i c  Is  both  and o b j e c t i v e s .  b i l i t y of n e o c o r t i c a l 2.  in  Such i n v e s t i g a t i o n s  r e l e v a n t t o the n e u r o l o g i c a l  objective  neuronal  measurements  the  effects. effects  neurons  to  of  applied  METHODS  Animals Duncan H a r t l e y  Animal  guinea  Care C e n t r e o f  the  pigs  (males  University  of  or  females)  British  were  Columbia.  obtained  from  The guinea  were weaned a t t h i s C e n t r e , from l a c t a t i n g f e m a l e s a f t e r 14 days,  the pigs  and f e d on  H. EL-BEHEIRY  a v i t a m i n C-supplemented chow, w i t h f r e e access  to water.  g u i n e a p i g s (150-250 g, o r a p p r o x i m a t e l y 20 days o l d ) Animal  Unit  Department  and of  experiment.  placed  in  Pharmacology  a  wire  cage  in  & Therapeutics  the  for  The t e m p e r a t u r e s and h u m i d i t y  Once a week,  5-6  were r e c e i v e d from the  animal  less  28  care  room  t h a n one week  in these f a c i l i t i e s  of  the  before  an  were c o n t r o l -  l e d a t 22-23°C and 50-55%, r e s p e c t i v e l y . 2.2  Slice  preparations  A n a e s t h e s i a was induced w i t h d i e t h y l e t h e r and m a i n t a i n e d by adminstration were  made  of  with  1-2%  halothane.  artificial  Attempts  ventilation  to  by  lower  endotracheal  intracranial  controlling  the  tension  end-tidal  CO,,  to  the  c o n c e n t r a t i o n at 30-35 mm Hg. After  frontotemporal  neocortex. and  other  In  the  the  derangements  uninterrupted and  view of  craniotomy,  of  cingulate  metabolism,  artificial  95/5% gaseous  cortices  constituents 1.25;  cerebrospinal  mixture of  the  MgS0 .7H 0, 4  The t i s s u e  2  of  2  2;  were  2  were  reflected  neocortical blood  (ACSF)  (pH 7.4) mM):  CaCl .2H 0  was trimmed i n t o ~5 mm square  that  within NaCl,  2;  2  tissue  immersed  in  continuously  was  brain  was  sensorimotor 1964;  Verne  oxygenated  10 s  of KC1,  dextrose,  10;  oxygenated  hypoxia  and immersed i n c o l d with  excision. 3.75;  a The  KH P0 , 2  NaHC0 ,  4  26.  3  glued with a-cyanoacrylate i n t o 500-600 nm  D u r i n g t h e s l i c i n g p r o c e d u r e which l a s t e d a t o t a l was  The  124;  blocks,  to  the  (Zeigler  t o a T e f l o n d i s c and c u t c o r o n a l l y w i t h a V i b r o s l i c e r slices.  of  excision.  identified  expose  neurons  supply  subcortical tissue,  (in 2  was  cortical  fluid  0 /C0  ACSF  the  of t h e  1974), e x c i s e d w i t h some c o n t i g u o u s (2°C)  dura  v u l n e r a b i l i t y of  up t o t h e moment  anterior  the  ACSF  at  thick  o f 15-20 m i n , t h e ~4°C.  The  slices  were t r a n s f e r r e d t o a chamber c o n t a i n i n g  oxygenated ACSF a t room t e m p e r a t u r e  (~22°C) and a l l o w e d t o warm up g r a d u a l l y  f o r a t l e a s t 30 min.  Each b l o c k  of  H. EL-BEHEIRY  t i s s u e y i e l d e d ~5 tical  neocortical  structures.  A  s l i c e was  volume, F i g . 1) c o n t i n u o u s l y atmosphere trolled  (Pandanaboina  a t 32-34°C  slices  together with  transferred  to  some c o n t i g u o u s  the  recording  1984).  with a thermoregulator.  Here,  the  subcor-  chamber  s u p e r f u s e d w i t h ACSF i n a h u m i d i f i e d  and S a s t r y  29  (1 ml  oxygenated  t e m p e r a t u r e was  con-  A n y l o n mesh p r e v e n t e d f l o a t i n g  movement of the s l i c e s d u r i n g p e r f u s i o n ( r a t e 2-4 m l / m i n ) . 2.3  S o l u t i o n s and drugs Althesin  (Glaxo),  and a l p h a d o l o n e diluted  on the  i n 20% v/v day  concentrations. these  which  of  the  absence  presence  mixture of  alphaxalone  (cremophor  to  obtain  EL),  the  2 x 10  El  -5  and  potencies  have  of  used  16 x 10  -5  alphaxalone  ascertained  that  the  1982, M i n c h i n 1981). was  vaporized  Fluotec  with  vaporizers  and bubbled  B e f o r e a p p l i c a t i o n by p e r f u s i o n , the  95/5%,  (Cyprane,  i n t o the ACSF  vapor  t h e chamber  also  atmosphere  was  gaseous  Keighley,  reservoirs  f o r a minimum of 20 min u s i n g Isoflurane  Og/^  UK)  in  were  isoflurane  observed  introduced  at  the  i m m e d i a t e l y above t h e  this  using  (Anaquest)  five  Mark  3  recently calibrated,  ( i n v e r t e d and c o v e r e d 60 ml  a d i l u e n t gas  the  C u l l e n and M a r t i n  mixture  that  in  % v/v.  a c t i v i t i e s o f A l t h e s i n on neuronal membranes are a t t r i b u t a b l e m a i n l y t o s t e r o i d , and not t o t h e v e h i c l e ( P e n n e f a t h e r e t a l . 1980;  was  desired  of t h e v e h i c l e i n t h e s o l u t i o n s  between  cremophor  steroids,  oil  e x p e r i m e n t w i t h the ACSF  on the a n a e s t h e t i c of  two  polyoxyethylated castor  ranged  investigations and  a 3:1  The c o n c e n t r a t i o n s  investigations  Previous  is  f l o w r a t e of designated submersed  1 L/min  syringes) (Fig.  concentration slice.  The  1). into  concen-  19 tration magnetic  of  isoflurane  resonance  in  the  bath  techniques  (cf.  was Miu  determined and  Puil  using 1989).  fluorine-nuclear The  molar  t h e r e b y o b t a i n e d were c o n v e r t e d t o t h e i r c o r r e s p o n d i n g MAC v a l u e s f o r  values rodents  Fig. 1 Anaesthetic delivery system and recording chamber used in these investigations.  co o  H. EL-BEHEIRY  (Steward  et  al.  1973;  Cullen  1986;  Firestone  et  ( - ) b i c u c u l l i n e m e t h i o d i d e (Sigma) and ( ± ) b a c l o f e n t h e ACSF from s t o c k Ca  2+  -deficient  2 mM C o C l  or  2  by  solutions  al.  1986).  (Ciba-Geigy)  Aliquots  of  were added  to  (0.1 mM) t h a t were kept f r o z e n u n t i l  solutions  were  increasing  the  31  required.  2+ made w i t h o u t Ca and e i t h e r by 2+ [Mg ] t o 4 mM, a c c o r d i n g t o t h e  adding exper-  2+  imental procedure. In s t u d i e s on t h e e f f e c t s of e x t r a c e l l u l a r [Mg ] 2+ ([Mg ] ) , t h e t o t a l d i v a l e n t c a t i o n c o n c e n t r a t i o n i n t h e ACSF was k e p t 2+ 2 c o n s t a n t by i n c r e a s i n g o r d e c r e a s i n g the [Ca ] wherever t h e [Mg ] was Q  +  changed.  The  osmolality  of  the  solutions  was  maintained  c o n s t a n t at 320 mosmol, by t h e a d d i t i o n o f sucrose where 2.4  I n t r a c e l l u l a r recording Intracellular  slice  in  the  recordings  recording  necessary.  arrangement  began a p p r o x i m a t e l y  chamber.  30 min a f t e r  A microelectrode  ^SO^.  The  a  filled  recordings  with  3M  resistances  from  KCl,  3M  measured  in  neurons Cs S0 , 2  the  4  were 3M  obtained  K-acetate  tissue  were  50  The e l e c t r o d e s were p u l l e d from m i c r o f i l a m e n t - i n c o r p o r a t e d g l a s s and  1.2 mm e x t e r n a l  (Narashige  diameter;  AP 81) u s i n g  was c h e c k e d u s i n g  ring  electrolyte  WPI  with  solution.  the  Instruments)  h i g h heat and magnet-power  high m a g n i f i c a t i o n  and p r o j e c t e d t i p s i z e . retaining  dissecting  . voltage  tip  using  Narashige  a p p r o x i m a t e l y at the m i d d l e and l o w e r t h i r d p o r t i o n of t h e  microelectrodes  guidance  a  microscope  Intracellular  visual  on  a  was  s l i c e ( c f . F i g . 2a)  under  mounted  incubating  micromanipulator, (40x),  positioned  approximately  light  with  a  settings.  microscopy  for  using  or to  0.6 M 90  blanks  vertical  Mft. (1.0  puller  Each e l e c t r o d e its  overall  form  The e l e c t r o d e s then were v e r t i c a l l y p o s i t i o n e d on a basal  end  immersed  overnight  in  the  concentrated  H. EL-BEHEIRY  32  Glued Iontophoretic  Electrode 20°  Electrode /  Both Slice  F i g . 2 Diagrams showing the neocortical s l i c e and s i t e s of recording stimulation (a) and types of iontophoretic electrodes used (b and c ) .  and  H. EL-BEHEIRY  The  input  resistances  intracellular circuit  i n j e c t i o n s of  o f a WPI  elicited  (R^)  at  of  the  neurons  specified  step  currents  a m p l i f i e r (model  0.1 Hz  by  M701).  intracellular  A series  injections  d u r i n g , and a f t e r an a n a e s t h e t i c a p p l i c a t i o n ; to 6 spikes.  A total  each e x p e r i m e n t a l amplitudes duration  and can  continuous  of  6 long-lasting  condition.  durations.  be  changes i n V .  by  subpial  region  of the  thetics  on s y n a p t i c  AHP  bipolar  potentials  unit  amplified  (VCR  potentials  adaptor; Medical  tape r e c o r d e r  (Canon model  computer.  some c a s e s  In  in  resting  voltage  sweep  into  slice  were  for  digital-to-analogue or  reproduction  Fig.  on  paper  in  pulses  evoked 5  their  and (V )> m  induced  placed  in  the e f f e c t s of  the  anaesset  to  V.  with  a  and s t o r e d  PCM-1  instrumentation  w i t h a super  f o r o f f - l i n e playback (in  VHS  video  and a n a l y s i s  analogue  form)  permits  for  an  plotter  manual  display  control  on t h e  by were  which d i g i t i z e s t h e  and s t o r a g e pen  peak  potential  S t i m u l a t i o n f r e q u e n c y was  thereby  for  amplitude  e l e c t r o d e was  ( B i o m a t i o n 805)  X-Y  as  membrane  i n t r a c e l l u l a r signals  and  before,  input of  the  oscilloscope,  (Hewlett-Packard  7015B;  3).  2.5  Computer Intracellular  were  conversion  and AHPs were  such  studying  digitized  VR-HF 600)  2048 p o i n t s  spikes  f o r any a n a e s t h e t i c  stimulating  ( F i g . 2a).  a n a l y z e d w i t h a waveform r e c o r d e r  bridge-balance  current  parameters  Systems I n c . )  the  the  each of t h e s e p u l s e s  0.2 Hz and the s t i m u l u s s t r e n g t h ranged from 5-10 The  using  AHPs were sampled c o n s e c u t i v e l y  changes  neocortical  of  of  were used t o compensate  A tungsten  m  via  measured  were q u a n t i f i e d by a v e r a g i n g  Because  affected  DC-injections  These  were  33  fed  into  an  analysis voltage  signals,  interface  panel  p l a y e d back from the VHS of  a  data  acquisition  l a b o r a t o r y - b u i l t ) w h i c h c o n v e r t s the i n p u t analogue  signals  video  recorder,  system  (ERAT;  t o d i g i t a l form  Stimulator  Sunning ^Amplifier  f v  DC Bias  VCR  *  WPI Anplif ier  •scilloscope c  „ PCM-1  Interface ERAT /  WPI Probe  Biomatlon  MINC  23 Terninal  /  XY  Fig. 3  Plotter  PDP 11/44  Organization of the experimental set-up for data display, acquisition and retrieval.  H. EL-BEHEIRY  (A/D  conversion).  MINC-23 computer of t h i s phase,  The (DEC  data  were  Inc.)  stored  during  temporarily  the d a t a  in  the  acquisition.  the f i l e s were t r a n s f e r r e d and  stored  memory  After  f o r the  o t h e r p r o c e d u r e s i n a n o t h e r computer (PDP 11/44; DEC I n c . )  35  of  a  completion  averaging  and  w h i c h has a l a r g e r  memory c a p a c i t y ( F i g . 3 ) . Fifteen  to  twenty  individual  traces  of  raw d a t a were s e l e c t e d from a MINC f i l e file  was  subsequently  duration,  created for  the  synaptic  transients  f o r an a v e r a g i n g  procedure.  the averaged  traces,  r a t e o f decay o f t h e s y n a p t i c t r a n s i e n t s  and  the  in  the  A new  amplitude,  were measured from t h e s e  averaged t r a c e s . 2.6  Iontophoretic 2.6.1  Electrodes.  electrodes  with  ultraviolet 358; at  (u. v.)  angle  Schwartz was  a  L o c t i t e U.K.  an  techniques  of  1967;  In the i n i t i a l  common  tip  sensitive  The  20-30°  and  an  Zieglgansberger at  'hockey-stick'  the  9-12 nm  The  and  Puil  a point  1973).  0.5  -  1 cm  of  substances  bond  the  with  or  70-90 Mn)  iontophoretic tip  at  the  hockey  stick  was  material  was  applied  (Caulk  Inc.).  a rapidly  tips.  This  setting assembly  parallel and  to  pipette  the  subjected  Secondary  ensured  wax  the  plane)  recording to  u.v.  strengthening  ( F i g . 2b)  that  of  horizontally  a g l a s s tube a c r o s s  dental  and  an a n g l e  positioned  an  Loctite  30-60 \im ( K r n j e v i c  The  shaped p i p e t t e then was  bonding  by a p p l y i n g from  of  ~4 mm from the  o f the e l e c t r o d e assembly was a c h i e v e d by g l u i n g or  glued  ( L o c t i t e glass  i n t e r t i p distance  l i g h t f o r ~3 min w i t h a Nuva L i t e gun  electrodes  were  (one i n the X-Y p l a n e and t h e o t h e r i n t h e X-2  'blade'  microelectrode.  of  L t d . ) t o the recording e l e c t r o d e s ( r e s i s t a n c e s ,  w i t h two m i c r o s c o p e s such t h a t  diameter  f l u i d adhesive  bent w i t h a m i c r o f o r g e  20-30°.  experiments, 5 b a r r e l l e d iontophoretic  t h e two at  about  transmitter  would be a p p l i e d i n t h e v i c i n i t y o f t h e soma o f an i m p a l e d neuron.  H. EL-BEHEIRY  In were  later  experiments,  separate  used i n c o m b i n a t i o n w i t h TTX  nearby  neurons  position neuron,  recording  and  application  to  iontophoretic eliminate  a c t i v a t e d by an e j e c t e d t r a n s m i t t e r  the i o n t o p h o r e t i c e l e c t r o d e t i p i t was  as  the  effects  substance.  In  possible  t o an  on  order  to  impaled  neuron  was  located  d i s t a n c e of ~200 um from the s u r f a c e of the s l i c e .  Since  the  iontophoretic  was  assumed t h a t t h e  near  electrodes  impaled  electrode  initially  as  36  positioned  at  an  angle  of  20°  with  the  horizontal  at  plane,  t r i g o n o m e t r i c f u n c t i o n s were used t o determine the amount o f t r a v e l f o r iontophoretic  electrode  into the  slice  (cf.  F i g . 2C).  This  twin  Once  a  neuron  was  applied current  (~80  slowly  independent  was  w i t h an  nA);  impaled,  Glu  was  ejected with  a  until  a maximal  the  continuously  the " a p p l i c a t i o n e l e c t r o d e " t h e n was advanced micromanipulator  the  electrode  placement p e r m i t t e d a p p l i c a t i o n s of the t r a n s m i t t e r s t o t h e d e n d r i t e s of neurons.  a  very  depolarization  observed. 2.6.2  Solutions  and equipment.  Four  barrels  a 32 gauge needle w i t h t h e f o l l o w i n g s o l u t i o n s a c e t y l c h o l i n e Cl  ( 0 . 5 M, pH 4 ) ,  pH 9 ) , GABA ( 0 . 5 M, pH 3.5)  f o r the e j e c t i o n currents;  as a c o n t r o l f o r e f f e c t s o f c u r r e n t s on The IDS  100 m i c r o i o n t o p h o r e s i s  two main f e a t u r e s .  It  c o u l d be passed t h r o u g h "drug-current"  and  this  had  from S i g m a ) : NMDA (200 mM,  50 mM, pH 3 ) .  b a l a n c i n g channel  u n i t used i n t h e s e  the N a C l - c o n t a i n i n g it  also  The  served  use  of a  a  barrel  built-in  experiments  possessed  'push-pull'  technique  i n magnitude  and  simultaneously sequencer  by  duration with  the  which  the  t r a n s m i t t e r s u b s t a n c e s c o u l d be a p p l i e d s e q u e n t i a l l y i n a p a t t e r n a t (preset)  using  ( N a C l , 10 mM) w h i c h was used  i n p o l a r i t y and equal  secondly,  filled  neurons.  a l l o w e d the a u t o m a t i c  i n which a c u r r e n t o p p o s i t e  (1 M, pH 8 . 5 ) ,  (Ciba-Geigy;  f i f t h b a r r e l was f i l l e d w i t h a c o n t r o l s o l u t i o n t o compensate  were  of drugs (mostly  Na S - g l u t a m a t e  and ( ± ) b a c l o f e n  usually  desired  c u r r e n t s f o r c e r t a i n d u r a t i o n s and i n t e r - a p p l i c a t i o n i n t e r v a l s .  H. EL-BEHEIRY  2.6.3  Experimental  assessed using (1)  after  the f o l l o w i n g stable  c u r r e n t was that  adjusted  responses of  evoked  conditions  i n the  range  ( ED50) o r  with a transmitter  The  effects  of  anaesthetics  were  procedures:  recording  a submaximal  (2)  procedures.  37  were  of  achieved,  30-350 nA  a just-maximal  the  iontophoretic  (typically  response  80  could  nA)  be  such  elicited  substance; r e l a t i v e l y constant  at a p p r o p r i a t e  intervals  amplitude  (10-30s)  for  (usually  a period  10-20 of  at  mV)  were  least  5-8  min; (3)  e q u i - a m p l i t u d e responses t o a t l e a s t two t r a n s m i t t e r s u b s t a n c e s  were  evoked i n t h e same neuron; (4)  perfusion  of  the  anaesthetic  was  commenced  during  the  continuing,  i n t e r m i t t e n t t r a n s m i t t e r a p p l i c a t i o n s , and (5)  at  just  least  3 or  before  4  responses  anaesthetic  anaesthesia,  and  quantitative  comparisons.  A  slightly  responses phylaxis  during  different  because  of  the  to  each  transmitter  application, the  recovery  procedure rapid  i n the l a r g e p y r a m i d a l  was  period,  used  development (layers  after  IV-V)  given were  for  of  substance  obtained  intervals  averaged  for  investigation  desensitization  neurons  (cf.  layers  of  of or  the  NMDA tachy-  11-111  in  2+ Thomson,  1986)  just-maximal short  to  a  low  dose  of  NMDA  e f f e c t was d e t e r m i n e d u s i n g  duration.  Then,  3 or  4  in  the  presence  infrequent  equi-amplitude  of  [Mg  ] .  The  (~3 min) a p p l i c a t i o n s  r e s p o n s e s were  obtained  of  at  3  min i n t e r v a l s b e f o r e commencing a n a e s t h e t i c a p p l i c a t i o n f o r ~9 min. 2+ 2.7 I n t r a c e l l u l a r Ca measurement 2.7.1 Wistar  rat  Neuronal embryos.  cultures. Cells  Hippocampi were d i s s e c t e d out from  were  dissociated,  put  onto  18-day-old  18mm c o v e r s l i p s  and  H. EL-BEHEIRY incubated  for 2  weeks  in  5% C 0  at  2  37°C  in  Dulbecco's  which c o n s t i u t e d 6 gm/L d e x t r o s e , 10 mM HEPES and 10% 2.7.2 Probes  Storage  Inc.)  and l o a d i n g  was  dissolved  p i p e t t e d i n t o small f o r 3 hours.  The d r i e d  stock  preincubated of  10 uM  Fura-2.  NaCl,  HEPES,  10;  EBSS  117; CaCl .H 0,  standard  the w e l l  EBSS;  Fura-2  solution  aliquots  sulfoxide  2  4  4 <  a  concentration  red  0.8;  2  1;  2  cell  and i n c u b a t e d f o r 2 hours.  (DMSO)  was added t o 2 ml  MgS0 H 0,  NaH P0 .H 0,  were  On t h e day o f t h e  (10  mM:  which  and vacuum-dried  (EBSS) y i e l d i n g  in  Molecular  K S0 , 2  dextrose,  indicator  , 5.6;  w i t h 1 ml  c u l t u r e then was p l a c e d f a c e - u p  Before  recording,  i t i n t o a separate  dish  4  (1 ml/L EBSS).  was immediately added t o t h e w e l l  t h e hippocampal  c u l t u r e was r i n s e d by p l a c i n g  AM (1 mg;  at -80°C.  and 0 . 5 % phenol  Fura-2  Fura-2  medium  serum.  i n 50 uL d i m e t h y l  Stock  26;  1.8  2  horse  and 50 LIL  stored  constituted 3  One ml o f E B S S - c o n t a i n i n g of  salt  NaHC0 ,  2  were  o f ~1 mM.  modified  placed i n a dessicator  was d i s s o l v e d  balanced  AM.  chloroform  aliquots  solutions  Earl's  2.7;  in  p l a s t i c ampules,  e x p e r i m e n t 50 ug o f F u r a - 2 yielding  o f Fura-2  38  in  the Fura-2  incubated  of standard  EBSS and  i n c u b a t e d a f u r t h e r h a l f - h o u r a f t e r which the c e l l s were ready t o use. 2+ 2.7.3  Intracellular  dye-loaded  cells  recording  chamber  solution,  low c h l o r i d e  isethionate,  were  Ca  inverted  allowing EBSS  2  measurement. and  sealed  perfusion  i n which  favoured  into  inputs  NaCl  a --  Stimulation by  preparation (Quis)-subtype  adding of  the  of  the  one  for  was s u b s t i t u t e d  of t h e t r a n s m i t t e r  NMDA-receptor  glycine  (1-2  LIM) and  low  chloride  EBSS.  subtype exclusion  by of  Activation  o f r e c e p t o r was a c h i e v e d by u s i n g  containing  specifically the  designed control  f o r 117 mM Na  and t h e o t h e r f o r a d m i n s t r a t i o n o f a n a e s t h e t i c s .  was used f o r t h e i n j e c t i o n of u l - v o l u m e s agonists.  Cover-slips  A third  port  substances  and  glutamate  was  Mg of  during  the  quisqualate  low c h l o r i d e EBSS  without  H. EL-BEHEIRY  t h e e x c l u s i o n o f Mg.  Continuous  s u c t i o n was a p p l i e d t o an o p p o s i t e  and the t e m p e r a t u r e was c o n t r o l l e d by a r e g u l a t e d h e a t i n g  39  opening  d e v i c e beneath the  chamber. The  cells  were  Jenalumar Z e i s s for  viewing  single and  under  epifluorescent  single  neurons  neurons.  oil  immersion  microscope Recording  at  which  of  lOOx  had  magnification  an  adjustable  fluorescence  from  light.  through  a  Fluorescence  G125  glass  signals  barrier  (emitted  filter  to  from  reduce  DC-voltage.  These  signals  were  c o n v e r t e r and s t o r e d on f l o p p y d i s c s presented  as  two  values  Ca-free-Fura-2  and  respectively.  The  converted  Ca-bound-Fura-2 2+ [Ca  digital  the at  ].  intensity  of  wavelengths  of  then  was  of  of  350  neuron)  was  background  the  fluorescence  form  w i t h a microcomputer.  representing  actual  to  a  a  aperture  perikarya  any  f l u o r e s c e n c e and then t o a p h o t o m u l t i p l i e r w h i c h c o n v e r t e d into  on  was c a r r i e d out at a l t e r n a t i n g e x c i t a t i o n wavelengths  380 nm u.v.  passed  viewed  by  an  The r e s u l t s emissions 380  and  calculated  of  A/D were both  350 nm,  using  the  following formula: CCa *],. . K 2 . 8 2  d  where is  the  rate  B(R-R )/«W  (1)  R)  min  constant  of  association  of  the  Fura-2  with  the  2+ c y t o s o l i c f r e e Ca 8 is  the  ,  r a t i o of  e x t r a c e l l u l a r Ca  2+  fluorescent  values  c o n c e n t r a t i o n ([Ca  2+  a t 380  nm w i t h z e r o and  ] ), Q  R i s t h e e x p e r i m e n t a l l y determined r a t i o of t h e the c e l l R  min  R  niaA m a v  and  1 S  is  infinite  values  of emission  from  a t 350 and 380 nm, t  n  e  r  the  a  t  l  °  °f v a l u e s at 350 and 380 nm a t z e r o [Ca  ratio  of  values  at  350  and  380  nm  at  2+ ] , Q  infinite  [Ca  2+  ] . 0  H. EL-BEHEIRY  B  '  R  min  2.7.4  a  n  d  R  max  a  r  40  determined from c a l i b r a t i o n o f t h e system.  e  C a l i b r a t i o n of  the  system.  The  following  calibration  procedure  was employed: Two hippocampal Ca -ionophore  Fura-2  A 23187  loaded c e l l  c u l t u r e s were  equilibrated with  the  the f i r s t i n t h e p r e s e n c e o f 10 mM EGTA 2+ ( c a l c i u m c h e l a t o r ) and z e r o [Ca ] , and t h e second i n t h e p r e s e n c e of 2+ 2 normal [Ca ] and w i t h o u t EGTA. [Ca ] . measurements were obtained 2 +  (10 nM),  +  Q  from both c u l t u r e s  for  several  was  6,  R„,-„ mi n  reached.  The  cells until  and  R„,„ max  a  'plateau'  constants  then  of c o n s i s t e n t were  values  calculated  from  t h e averaged v a l u e s o b t a i n e d i n these e x p e r i m e n t s . 2.8  Statistical  analysis  The e l e c t r o p h y s i o l o g i c a l r e s u l t s were s u b j e c t e d t o t e s t s w i t h ANOVA f o r statistical using  significance.  t h e Mann Whitney  was u s u a l l y  5 unless  was p<0.05.  Regression  3  M u l t i p l e comparisons  U-test  (Zar  otherwise  1984).  stated.  between two groups were made  Sample  The  size  in  accepted l e v e l  all  experiments  of  significance  a n a l y s i s was used t o f i t t h e d a t a .  SPONTANEOUS ACTIVITY, REPETITIVE FIRING AND PASSIVE MEMBRANE PROPERTIES  3.1  Results The  64  parameters  neurons of  selected  neuronal  for  the  e x c i t a b i l i t y had  t h a n -60 mV, s p i k e a m p l i t u d e s u s u a l l y such s p i k e s  study  r e p e t i t i v e l y during  resistances  (Vm;  potentials  i n t r a c e l l u l a r current-pulse  cortex. potentials  - 7 1 . 6 * 9.8 mV)  applications more  on  negative  o f 70-75 mV, and an a b i l i t y t o f i r e  impalements corresponded  input  anaesthetic  resting  depths o f s u c c e s s f u l The  of  (mean * SD; remained  to layers  IV  injection.  for  The  and V of t y p i c a l  6 5 . 7 > 30.7 Mn) stable  5  periods  and of  resting 30  to  H. EL-BEHEIRY  240 m i n .  Forty-five  applications  of  isoflurane  0.5 t o 2.5 MAC were made t o 34 neurons. neurons,  in  doses  that  41  ranged  from  A l t h e s i n was a p p l i e d 48 t i m e s t o 30  i n c o n c e n t r a t i o n s from 15 nM to 1500 uM ( a l p h a x a l o n e  content).  The  v e h i c l e , cremophor EL d i d not have any e f f e c t when t e s t e d on t h e p a s s i v e a c t i v e membrane p r o p e r t i e s as w e l l as t h e spontaneous In  the  initial  investigations  100 uM A l t h e s i n were  evident  depending  was  at  on t h e  p e r m i t sampling  applied  on  6-9 min  with  rate  perfusion.  of  e f f e c t s was observed  (synaptic  on  percent  and  at  t o 10  The  maximal  4-6 min  the  isoflurane  with  rate  was  neurons.  neurons  that  have  been  or  effects Althesin,  adjusted  induced by e i t h e r  to  agent  No d i f f e r e n c e  between t h e d i f f e r e n t t y p e s o f  spontaneous  (i.e.,  transients  Applications  of  spontaneous  firing  activity  was  (Berg-Johnsen  not  of  evident,  and Langmoen  after  an  increase  even  and  after  as  in  firing.  about  30 min  10/12  classified  in  sensorimotor into  and  found  Full  induced changes were f u l l y r e v e r s i b l e .  were  spontaneously  three  by in  5/8  recovery  active spikes.  depressed  neurons).  inhibition hippocampal  firing.  fired  e f f e c t or  Althesin,  recovery  (Fig. 4 A).  repetitive  had no  Occasionally, Complete  evoked  arrhythmically  either  followed  also  1987).  application.  and  ( F i g . 4)  firing  and  the neurons  spikes)  (isoflurane,  potentiation  activity  16/64) o f  an a n a e s t h e t i c  initial  cases  18 m i n .  Therefore,  was a p p l i e d 18 t i m e s  cingulate  Effects  Twenty-five  rebound  for  1 MAC  neurons.  (McCormick and P r i n c e 1986).  3.1.1  6-10 min  neurons,  o f the maximal changes i n parameters  anaesthetic  categories  the  isoflurane  Halothane  anterior  of  continuously  at 6 m i n .  and  8  a c t i v i t i e s of  and  of  spontaneous  neurons  in v i t r o  u s u a l l y was observed recovery was  However,  not most  was  within  preceded  observed other  An  in  by  a  some  anaesthetic  H. EL-BEHEIRY  IILI11L. Isoflurane  HMACI  42  jiunN  HULL  Jin  i  ni  Isoflurane I2.SMACI  A l t h e s i n I100/1MI  5mV imin  B  control  IFLIMAC  20 mV 0.5nA  recovery  LA  Fig. 4 I s o f l u r a n e and A l t h e s i n i n d u c e d d e p r e s s i o n o f spontaneous spike activities. (A) i s o f l u r a n e a p p l i c a t i o n (1 MAC; upper trace) produced i n h i b i t i o n o f t h e spontaneous f i r i n g and a rebound i n c r e a s e i n f i r i n g a f t e r Isoflurane ( 2 . 5 MAC; m i d d l e t r a c e ) i n t h e same neuron the a p p l i c a t i o n , produced a s l i g h t h y p e r p o l a r i z a t i o n (~3 mV) and a d e p r e s s i o n o f spontaneous a c t i v i t y t h a t was p a r t l y r e v e r s i b l e . A l t h e s i n (100 LIM; l o w e r t r a c e ) i n d u c e d ~5 mV h y p e r p o l a r i z a t i o n a s s o c i a t e d w i t h complete a t t e n u a t i o n o f spontaneous (B) on a f a s t e r time b a s e , isoflurane activity i n a second neuron. d e p r e s s e d spontaneous s p i k e s and b l o c k e d t h e AHPs. V o l t a g e responses t o h y p e r p o l a r i z i n g c u r r e n t p u l s e s may be seen a t the l e f t o f the anodal break spikes. Complete r e c o v e r y was o b s e r v e d a f t e r 10 m i n . There was no change i n t h e V o f t h i s neuron. m  H. EL-BEHEIRY  The s i n g l e not  altered  s p i k e t h r e s h o l d t e s t e d i n 62 neurons significantly  in  (~70%) o f  c u r r e n t r e q u i r e d t o evoke 3 t o 5 s p i k e s spikes  during  2.5 MAC  or  Althesin of  applications  Althesin  (17/30  repetitive  applications  neurons)  (9/32  firing  and 8/30  3.1.2  Effects  isoflurane  effect  was  Isoflurane tion  in  doses  of  e f f e c t i v e in producing  <2  of  neurons)  elicited  ( 1 - 2 . 5 MAC)  8.8 * 2 . 5 % i n applications  reductions  in  at 1.5,  in  were  these  doses  ~46% from  control  values  1500 uM ( c f . F i g . 7D). ane ( 1 . 5 MAC) R.j  in  9  Na -spike +  as  genesis.  inhibition  and  properties.  of  34  cells  at  2  and  dose  5 neurons.  in  doses  Althesin neurons  to the  Applications 3-8 mV.  2.5 MAC  The  of  resistance  This  (Fig.  7A).  hyperpolariza-  hyperpolarization  more  (R^)  2 and 2.5 MAC ( F i g .  15.34%,  was  (100 nM)  observed  by  evoked a mean  than  2 0 . 7 1 % and  observed  with  produced  previously  in  were  7B).  28.53%,  150 uM  observed  The mean  was  with  decreases  respectively.  A maximal  Althesin  Concomitant a p p l i c a t i o n of TTX  or A l t h e s i n  neurons  to  5A).  i n j e c t i o n through  e f f e c t of 1.5 MAC i s o f l u r a n e on R^ i s shown i n F i g . 8. of  1  (Fig.  produce  the a b i l i t y of  of  7B). input  isoflurane applications  24  only  at t h e h i g h e s t  s t a t i s t i c a l l y significant (Fig.  at  doses  isoflurane  electrical  significant  of  Althesin  Significant  in  100-1500 uM  to d i r e c t c u r r e n t pulse  hyperpolarized  administrations  with  compromised  on Vm and membrane  control)  Hence,  the  6A-B).  statistically  (from  R.j  amount  (23/32 neurons)  ( F i g . 5B).  r e p e t i t i v e l y i n response  recording electrode (Fig.  of  isoflurane  However,  a d m i n i s t r a t i o n a t the 15 o r 50 nM doses d i d not  evoked  fire  of  by c u r r e n t i n j e c t i o n was  cases.  was o n l y  43  reduction  applications  (1.5 uM) and  at  isoflur-  the  same e f f e c t s on t h e V  the  absence  of  The  m  TTX-blockade  and of  H. EL-BEHEIRY  44  Fig. 5 A n a e s t h e t i c - i n d u c e d increases in the t h r e s h o l d f o r spike genesis. (A) u s i n g same c u r r e n t i n t e n s i t i e s , i s o f l u r a n e (IFL 1 MAC) and A l t h e s i n (AL 100 uM) decreased t h e number of s p i k e s evoked i n two s e n s o r i m o t o r neurons. Note the d e p r e s s i o n of t h e a m p l i t u d e o f the second s p i k e and the p o s t s p i k e AHP d u r i n g a n a e s t h e t i c a p p l i c a t i o n . was h e l d c o n s t a n t t h r o u g h o u t t h e experiment. (B) no d e c r e a s e i n r e p e t i t i v e f i r i n g o c c u r r e d but a r e d u c t i o n i n the AHPs was e v i d e n t d u r i n g A l t h e s i n a p p l i c a t i o n (50 nM). Complete r e c o v e r y was observed a f t e r 9 m i n . S p i k e s are t r u n c a t e d due t o f r e q u e n c y response of p e n w r i t e r r e c o r d e r . V d i d not change d u r i n g the e x p e r i m e n t . m  IFL 1 MAC  -MM  r e c o v e r i e s  JJ AL 100pM  llOmV 50ms  _J  B  m.. J  AL 5 0 M  c o n t r o l  L  r e c o v e r y  M  m  10  UM _ n2nA —i  75  m s  mV  oo  5 4^ cn  H. EL-BEHEIRY  Recovery  IFL1MAC  Control  46  AAAJ  s  J  J  1nA  Recovery 15 mV  _r  F i g . 6 Anaesthetic interference with the r e p e t i t i v e f i r i n g a b i l i t i e s of n e o c o r t i c a l neurons. (A) i s o f l u r a n e ( I F L 1 MAC) i n t e r f e r e d w i t h r e p e t i t i v e f i r i n g e l i c i t e d by c u r r e n t p u l s e i n j e c t i o n s . When t h e i n j e c t e d c u r r e n t was i n c r e a s e d ( l o w e r t r a c e s ) , t h e a b i l i t y o f t h e neuron t o f i r e more than 1 l a r g e a m p l i t u d e s p i k e s t i l l was s e v e r e l y compromised. The AHPs a l s o were reduced. (B) A l t h e s i n ( A L ; 75 M) depressed t h e r e p e t i t i v e f i r i n g a b i l i t i e s o f an a n t e r i o r c i n g u l a t e neuron. V,^ i n A and B were c o n t r o l l e d a t t h e r e s t i n g l e v e l s ( ~ -74 mV). S p i k e s a r e t r u n c a t e d due t o f r e q u e n c y response o f penwriter recorder. M  151  B  ' i z a t ion  15  10  10  Hypei•pol  ra  o a.  w 01  a >» X  0.4  1  2  5  10  Isoflurane concentration IMACI  1 2 Isoflurane concentration IMACI  100  1000  Althesin concentration IjiMl  3  10  100 1000 Althesin concentration f MI M  m i—i  73 -<  F i g . 7 A l t e r a t i o n s i n t h e p a s s i v e membrane p r o p e r t i e s by a p p l i c a t i o n s o f i s o f l u r a n e (A,B) and A l t h e s i n (C,D). Symbols ( • ) i n d i c a t e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e from mean c o n t r o l v a l u e s a t p<0.05.  ^ ^  H. EL-BEHEIRY  48  F i g . 8 E f f e c t s o f i s o f l u r a n e (1.5 MAC f o r 6 min) on t h e i n p u t r e s i s t a n c e o f an a n t e r i o r c i n g u l a t e neuron. (A) responses t o i n j e c t i o n s o f c u r r e n t p u l s e s in c o n t r o l , i s o f l u r a n e and r e c o v e r y c o n d i t i o n s . Note t h a t isoflurane a p p l i c a t i o n produced a s l i g h t decrease i n i n p u t r e s i s t a n c e . The t h r e s h o l d amount of c u r r e n t f o r e l i c i t i n g one s p i k e was i n c r e a s e d . (B) t h e s l o p e r e s i s t a n c e was d e c r e a s e d by ~28% d u r i n g i s o f l u r a n e a p p l i c a t i o n . Complete r e c o v e r y was o b s e r v e d .  H. EL-BEHEIRY 3.2  Discussion The  the  spontaneous  level  for  stimuli  (Krnjevic  spontaneous cant,  a c t i v i t y of  receiving,  actions  conductance  of  dose  1983).  reacting  1983).  been c o r r e l a t e d  This  level  to  The  internal observed  V  and  m  isoflurane  R^,  suggests  and A l t h e s i n  observed  When  by a n a e s t h e t i c  with  low doses  the  of  optimally  and  external  depression  actions  that  at  Consequently,  another  possibility  conductance  may  be  some  low d o s e s )  the  neuronal  applications were  could  transmitter  considered  about  For  example,  Bosnjak  the  applied,  the  been  release. m  the  partly  Addition  or  R...  failure  in  Whitney  the  of  recording  anaesthetics  investigations  excitability anaesthesia  and Glenn 1986;  Takenoshita  synaptic inputs  the  1987)  and  in  to  actions  electrode  which  T h i s can e x p l a i n c e r t a i n i n c o n s i s t e n c i e s  effects some  to  in  in  have  where  have  on  not  of  revealed  the  postsynaptic  Berg-Johnson peripheral 1987).  membrane (Nicoll  and Langmoen sensory  neurons  1987;  in  membrane changes  in  Sc hoi f i e l d  Nonetheless,  i n some c e n t r a l neurons  ( P u i l and Gimbarzevsky  passive  ( Z o r y c h t a and (Jfapek 1978;  e t a l . 1982 and F u j i w a r a e t a l . 1988).  demonstrated d u r i n g  and  the  distal  e x c i t a b i l i t i e s o f CNS neurons  reductions  1982;  sites  somatic i n l o c a t i o n .  literature  properties.  1980;  at d e n d r i t i c  of  sites.  observe a change i n c o n d u c t a n c e would be a t t r i b u t a b l e t o a n a e s t h e t i c  was l i k e l y  of  s i g n i f i c a n t increases  anaesthetics  decrease  least  o f TTX, however, d i d not change t h e e f f e c t s of a n a e s t h e t i c s on V  (in  to  is  at p r e s y n a p t i c  anaesthetic  the  i n membrane  that  are  a c t i o n s are i n d i c a t e d because  were  range.  in  e x t e n t of t h e changes  masked  and  Richards  changes  However, p o s t s y n a p t i c  full  (Webb  has  a c t i v i t y i n the s l i c e p r e p a r a t i o n t h a t o c c u r r e d w i t h o u t s i g n i f i -  observed  higher  analyzing  1974b;  concomitant  input  n e o c o r t i c a l neurons  of a c t i v a t i o n i n t h e b r a i n  required  the  49  profound have  and  been  Madison  Takahashi  without  known  H. EL-BEHEIRY  The r e p e t i t i v e f i r i n g by  the  anaesthetics,  change  in  local  especially  Na -inactivation  diminutions  tion.  in  +  anaesthetic-like  observed  evoked by c u r r e n t isoflurane. the on  neuron  the  injections  This  was  e f f e c t could  (Hodgkin  Na -channels  and  o f t h e evoked  depressed be  Huxley  seems  +  i n the a m p l i t u d e and shape  i n most neurons  In  effect  pulse  50  due  to  1952).  A  unlikely  single  spikes  because were  d e s p i t e changes i n t h e r a t e o f c u r r e n t p u l s e  a d d i t i o n , the f i r s t s p i k e  the anaesthetic a p p l i c a t i o n s  i n an evoked b u r s t  (Fig. 6 A-B).  was  not  not  injec-  a f f e c t e d by  The compromised r e p e t i t i v e f i r i n g  a b i l i t y can be a t t r i b u t e d t o an a l t e r a t i o n i n t h e l e v e l of N a - i n a c t i v a t i o n +  as  a r e s u l t of anaesthetic  attenuations  of the AHPs ( c f .  Figs.  4B,  5 and 6  2+ and  Section  VII)  secondary  ( K r n j e v i c and P u i l cal  neurons  1988)  to f i r e  In  this  series  and A l t h e s i n neurons of  (10  showed  in  - 200 uM) (iSEM)  resting  Mn.  They  fired  with  overshoots  (EPSP)  Ca  -currents  potentials  of  of  ( 0 . 5 - 2 . 5 MAC)  on 37 neurons.  -74 * 4 mV and  spikes  ~20 mV.  (<2 ms The  in  average  recordings  KC1 or  (3 M) Cs S0 2  4  potentials  for  the e x c i t a t o r y  (3 M)  for  the  These  a mean  R.  duration)  of  duration  of  The e l e c t r o d e s were f i l l e d w i t h K - a c e t a t e  s t u d i e s on i n h i b i t o r y p o s t s y n a p t i c 4.1  were observed  repetitive  on c e r t a i n o t h e r o c c a s i o n s ,  potentials  inward  the e f f e c t s o f i s o f l u r a n e  applications  r e c o r d i n g was 125 (±78) min. and,  of  EFFECTS ON SYNAPTIC TRANSIENTS  of i n v e s t i g a t i o n s  amplitude  depression  r e p e t i t i v e l y ( S t a f s t r o m e t a l . 1985).  mean  65.9 * 3.65  >70 mV  a  which p a r t l y c o n t r i b u t e t o t h e a b i l i t y o f n e o c o r t i -  spikes 4  to  (2 M)  postsynaptic dose/response  (IPSPs).  Results 4.1.1  Resting  continuously  on  a  membrane pen  properties.  recorder  whereas  In R.  this was  series, monitored  V  m  by  was  monitored  injection  of  H. EL-BEHEIRY  hyperpolarizing  pulses  r u p t i n g the rhythmic  in  between  two s u c c e s s i v e  s t i m u l a t i o n (0.2  (2.5 MAC)  only  elicited  An  isoflurane-induced  a t doses >1.5 MAC.  hyperpolarized  3  by A l t h e s i n were  neurons  not  A p p l i c a t i o n at  by  3-5 mV.  The  in  R^  were  2 . 5 MAC.  In  25% a f t e r  8 min  observed  3 neurons, of  during  inter-  isoflurane  (2.5 MAC).  hyperpolariza-  the highest  t h e 200 nM  Statistically  significant  applications  No  dose  even a t  the maximum mean d e c r e a s e  application  by  hyperpolarizations  statistically significant  dose where 4 neurons were h y p e r p o l a r i z e d by ~4 mV. changes  or  Hz) j u s t b e f o r e , a t t h e end, and a f t e r  ~12 min o f an a n a e s t h e t i c a p p l i c a t i o n . t i o n was observed  stimulations  51  at  1.5,  from c o n t r o l  significant  2  and  values  was  changes  in  _R.  were o b s e r v e d d u r i n g A l t h e s i n a p p l i c a t i o n s at a l l doses (10-200 ^M). 4.1.2  Dose-response  (EPSPs). cal  Focal  using  and l o w e r t h i r d  were  0.2 H z ) .  When  V ) the  resting  by c o n t i n u o u s mean  rate  portions  DC-current  rise mV/ms.  dependent neurons;  (±SEM) of  was  in  of  postsynaptic  (±30)  F i g . 9A).  an  stimulation  anaesthetic  constant  at  the  initial  ms  Avoli  and O l i v i e r  (cf.  mV/ms  of  whereas  isoflurane in  2 MAC  the the  a  the  rate  ( 1 - 2 . 5 MAC) amplitude  mean  of  depressions  v a l u e s were 52.56 and 8 0 . 2 5 % r e s p e c t i v e l y ( F i g . 1 0 ) .  action  decay  produced  from  level The  with  1989).  the  in  period.  (±0.55) mV  of  (at  change  resting  the e x p e r i m e n t a l 18.5  By  experiments  induced  was  of  slice.  EPSPs w i t h o u t  EPSPs  (±0.02)  1 and  sectioned  control  depression At  l o c a t e d at t h e j u n c t i o n  coronally  these  potentials  r e g i o n of t h e n e o c o r t i -  (4-15 V; 0.1 ms),  when  held  the  Application  reversible  the  ~50% of  was  cells  i n j e c t i o n throughout  210  0.25  of  stimuli  (i.e.,  potential  duration  of  (±0.01)  initiated necessary  amplitude  average  excitatory  EPSPs i n the p y r a m i d a l  constant j u s t - t h r e s h o l d  potentials  of  s t i m u l a t i o n i n the immediate s u b p i a l  s l i c e produced  the middle  depression  an  The  mean  was  0.07  a  dose-  EPSPs  (16/16  the  control  The r a t e s o f r i s e  of  H. EL-BEHEIRY  52  F i g . 9 E P S P - d e p r e s s i o n i n d u c e d by i s o f l u r a n e and A l t h e s i n . (A) i s o f l u r a n e ( I F L ) was a p p l i e d t o two s e n s o r i m o t o r neurons f o r 10 min i n doses of 1 and 2 MAC. The superimposed t r a c e s show almost complete d e p r e s s i o n a t 2 MAC. Recovery was e v i d e n t a f t e r 12 m i n . (B) A l t h e s i n 75 and 200 was a p p l i e d t o two a n t e r i o r c i n g u l a t e neurons f o r 8 min. P a r t i a l r e c o v e r y was o b t a i n e d a f t e r 15 min i n t h e second neuron ( l o w e r t r a c e ) .  H. EL-BEHEIRY  53  150 ms  IFL  1.5  MAC  B 100-r  0.4 0.5  1.0  2.0  Isoflurane (MAC)  0.4 0.5  1.0  2.0  Isoflurane (MAC)  F i g . 10 Dose-dependent s u p r e s s i o n o f t h e a m p l i t u d e and r a t e o f decay by i s o f l u r a n e ( 0 . 5 - 2 . 5 MAC), (A) computer-averaged t r a c e s o f EPSPs evoked i n a s e n s o r i m o t o r neuron d u r i n g c o n t r o l and i s o f l u r a n e ( I F L ; 1.5 MAC) p e r f u s i o n u s i n g t h e same s t i m u l u s s t r e n g t h and d u r a t i o n . T r a c e s averaged were o b t a i n e d over a p e r i o d o f 2 min b e f o r e , and a t t h e end o f isoflurane application (frequency o f s t i m u l a t i o n was 0.2 H z ) . (B) d o s e - r e s p o n s e r e l a t i o n s h i p s f o r t h e p e r c e n t d e p r e s s i o n o f a m p l i t u d e s , r a t e s o f r i s e and r a t e s o f decay o f t h e averaged EPSPs. The sample s i z e f o r each dose was >4. S t a t i s t i c a l s i g n i f i c a n c e (p<0.05) from c o n t r o l v a l u e s i s i n d i c a t e d by T*). Note t h a t d e p r e s s i o n o f t h e r a t e of r i s e was s i g n i f i c a n t a t 2 . 5 MAC. E r r o r bars r e p r e s e n t s t a n d a r d d e v i a t i o n s .  H. EL-BEHEIRY the  EPSP  whereas and  were  control  isoflurane,  conditions  respectively  were  neurons).  200 uM.  EC  (±3.67,  The  for  5 Q  to  at  concentrations  of  of  on EPSPs,  the  4.1.3 observed  prolongation  depression  1980),  of  of  the  or  bicuculline  of  percent  depression  observed  or  Cs S0 2  4  w i t h i n 5 min o f  increase  convulsant,  due  impinging  on  these  neurons  Under such c o n d i t i o n s , control, usually  IPSPs by  of  associated  (1.5 MAC) and A l t h e s i n  the  are  by  77.88  -  (n = 4  Althesin  in depressing  was  presumed  anaesthetic  to  at the  (±2.9)  with  at  Matsumura strength  EPSPs  initiated  w i t h 2-3  action  Because  the  contribute  to  agents  (Scholfield  isolate  of  the  a f l o w r a t e of during  of  et a l .  the  EPSPs  and A l t h e s i n .  blockade  observed  preponderance  (cf.  (0.002  was observed  IPSP-blockade.  F i g . 14C),  i n R^ was  the s t i m u l u s  just-threshold were  to  the  11).  bicuculline perfusion  No d e p o l a r i z a t i o n o r possibly  (cf.  all  conductance  used t o p h a r m a c o l o g i c a l l y  electrodes  from  occurred  in  EL  amplitude  f o r a more e x a c t i s o l a t i o n of the e f f e c t s of i s o f l u r a n e K-acetate  effects  cremophor  EPSP  0.5  ( ± 1 . 7 4 ) mV/ms  Althesin  potent than i s o f l u r a n e  produced  was  of  isoflurane  the  At  depressions  and complete s u p p r e s s i o n  allocortical  (50 uM)  effects  on the membrane  i n the presence  EPSPs  percent  vehicle,  depression  the mean maximum  EPSP a t t e n u a t i o n  the  the  above 50 uM ( F i g . 11)  decay;  mean  >2 MAC  manner.  S.E.M.) mV/ms and 47.74  that  However, A l t h e s i n was l e s s  of  the  c o n c e n t r a t i o n s as high as 200 uM ( F i g .  the  except  dose-dependent  Perfusion  had no e f f e c t  The  slightly  rates  affected  example,  18.8  similar  (n = 1 3 ) .  0.016% v / v ) ,  for  ( F i g . 10).  a time course  neurons  was  significantly  the r a t e s o f decay were reduced i n a dose-dependent  1.5 MAC  with  not  54  Using  IPSPs  2 ml/min.  a p p l i c a t i o n of  tonic 1988;  excitatory Buzsaki  epileptiform  potentials  ( F i g . 12A).  bursts  the  inputs 1984).  t h a t was r e q u i r e d t o e l i c i t only  was  the that  Isoflurane  (75 nM) a p p l i e d c o n c o m i t a n t l y w i t h b i c u c u l l i n e  H. EL-BEHEIRY  55  A  o -j 10  1  1  20  50  1 100  Althesin (/xM)  1 — 200  -| "—i 10  20  1  50  1 100  Althesin (/xM)  1—  200  F i g . 11 A l t h e s i n - i n d u c e d dose-dependent d e p r e s s i o n o f t h e a m p l i t u d e s and r a t e s o f decay o f n e o c o r t i c a l evoked EPSPs. (A) computer-averaged t r a c e s evoked i n an a n t e r i o r c i n g u l a t e neuron a r e shown b e f o r e and d u r i n g A l t h e s i n application (AL; 50 LIM). Each a v e r a g e d - t r a c e represents a t l e a s t 15 individual traces. (B) a m p l i t u d e s and r a t e s o f decay were s i g n i f i c a n t l y d e p r e s s e d i n a dose-dependent manner, whereas t h e r a t e s o f r i s e showed s t a t i s t i c a l l y s i g n i f i c a n t s u p p r e s s i o n o n l y a t t h e h i g h e s t dose (200 u.M). Symbols (*) i n d i c a t e s t a t i s t i c a l s i g n i f i c a n c e (p<0.05) from c o n t r o l . Sample s i z e s were 2. e x c e p t a t 15 and 200 LIM ( n = 3 i n e a c h ) . 4  H. EL-BEHEIRY  56  F i g . 12 Epileptogenic activity and EPSP suppression by anaesthetic a p p l i c a t i o n s i n the p r e s e n c e of GABAergic b l o c k a d e i n n e o c o r t i c a l neurons. (A) A p p l i c a t i o n of b i c u c u l l i n e (BIC; 50 MM) i n i t i a t e d e p i l e p t i c a c t i v i t y i n a neuron t h a t was responding t o the same s t i m u l u s w i t h an EPSP. (B-C) e p i l e p t i c b u r s t s induced by b i c u c u l l i n e i n two neurons were s u p p r e s s e d by the a p p l i c a t i o n of i s o f l u r a n e and A l t h e s i n ( I F L , 1.5 MAC, 15 min and AL, 75 nM, 12 min r e s p e c t i v e l y ) . In (B) t h e time c o u r s e of b u r s t s u p p r e s s i o n i s shown ( m i d d l e t r a c e , 5 min and l o w e r t r a c e , 12 m i n ) . (D) i n low Ca media an EPSP c o u l d be evoked during the concomitant application of bicuculline. Under such c o n d i t i o n s , a concomitant a n a e s t h e t i c application (~10 min) a t t e n u a t e d the EPSPs i n a s i m i l a r manner t o t h a t observed i n t h e absence of b i c u c u l l i n e ( c f . F i g . 9 ) . 2 +  H. EL-BEHEIRY  depressed (Fig.  the  amplitudes  12B-C).  In  of t h e e p i l e p t i f o r m b u r s t s ,  unmasking  order to e l i m i n a t e the a c t i o n p o t e n t i a l s  58  small  EPSPs  and e p i l e p t i f o r m  2+ a c t i v i t y , the e x t r a c e l l u l a r 2+ the  [Mg  ]  procedure,  was  Ca  increased  c o n c e n t r a t i o n was  by  EPSPs s u b t h r e s h o l d  an  to  equivalent  spike  decreased  amount.  genesis  could  neurons,  respectively),  the  EPSPs  were  be evoked.  Effects  Supramaximal  on  stimulation  anaesthetic  perfusion  50 uM doses  to  ( F i g . 13,  Al-Bl).  (isoflurane  On  spikes  were evoked  at  was  1.5 MAC  in these  for  evident  interrupting  the  neurons  spikes to  12 min  after  that  3  flow  ~15 min  subpial  r e v e a l e d t h e compromised  Dose-response  Cs-blockade  of  the  manner  on  spikes.  blocked  by  and A l t h e s i n  in  of  2 ml/min  returning  stimulation,  a b i l i t i e s of  relationship  K-conductances.  a  more  to  the  than  two  w i t h i n j e c t i o n s of d e p o l a r i z i n g  same number of s p i k e s as i n c o n t r o l c o n d i t i o n s 4.1.5  the  evoked  rate  The same c u r r e n t i n j e c t i o n i n t e n s i t i e s used d u r i n g  applications  After  in  were  neurons at  this  12D).  intracellular^  evoked  3 neurons)  Recovery  solution.  and  consistently  additional  control  pulses.  orthodromically  of  and 75 ^M i n 4  depressed  s i m i l a r t o t h a t observed i n the absence of b i c u c u l l i n e ( F i g . 4.1.4  1 mM and  Because  a p p l i c a t i o n of i s o f l u r a n e and A l t h e s i n (1.75 MAC i n 4 neurons additional  to  (Fig.  for The  the  use  the  neurons  current  anaesthetic to  fire  the  13A2-B2).  IPSP  suppression  during  of  intracellular  Cs S0 2  4  e l e c t r o d e s was a s s o c i a t e d a f t e r about 15 min of impalement (n = 17  neurons),  with  value  an  input  resistance  55.75 *3.25MQ t o a mean  of  which  increased  74.57  (*4.25) Mn , an average  ~11 mV and a markedly p r o l o n g e d C s - a p p l i c a t i o n the postspike ( F i g . 14B). neurons;  from  spike duration  mean  (Fig.  14A).  control  depolarization  point  blocked the  Thus t h e use of t h e C s S O . - c o n t a i n i n g 9  of of  A f t e r 20 min o f  a f t e r h y p e r p o l a r i z a t i o n was c o m p l e t e l y  A p p l i c a t i o n of b i c u c u l l i n e a t t h i s  F i g . 14C).  a  abolished IPSPs  electrodes  (4  H. EL-BEHEIRY  59  B2 I  AL 50 M M  F i g . 13 A n a e s t h e t i c i n t e r f e r e n c e w i t h t h e s y n a p t i c a l l y - i n d u c e d s p i k e s and directly-evoked action potentials by intracellular current pulse injections. S y n a p t i c a l l y - i n d u c e d ( A l ) and d i r e c t l y - e v o k e d s p i k e s (A2) were depressed by i s o f l u r a n e ( I F L ; 1 MAC) i n a s e n s o r i m o t o r neuron. ( B l and B2) the same e f f e c t was produced by Althesin (AL; 50 LIM) i n another s e n s o r i m o t o r neuron. C a l i b r a t i o n bars: 120 ms ( h o r i z o n t a l ) and 10 mV ( v e r t i c a l ) i n A l and B l , 100 ms and 40 mV o r 1 nA i n A2 and B2.  H. EL-BEHEIRY  Internal  111..  o  C s  +  -  60  application  15 min  3 min  50 ms  "J20mV  -70"  B  u 20 min  2 min  o.3s 10 mV  160ms  D BIC  5mV  F i g . 14 Internal Cs -application in 4 neocortical neurons. (A) Cs l e a k a g e from an i n t r a c e l l u l a r m i c r o p i p e t t e caused p r o l o n g a t i o n of the a c t i o n p o t e n t i a l s , p a r t i a l b l o c k a d e of t h e AHP and a small l o c a l a n a e s t h e t i c - l i k e e f f e c t i . e . , a r e d u c t i o n i n the r a t e s of r i s e and a m p l i t u d e s of the s p i k e s a f t e r 15 min o f impalement. (B) i n another a n t e r i o r c i n g u l a t e neuron complete blockade of the AHP was e v i d e n t a f t e r 20 min of p e n e t r a t i o n . In (A) and ( B ) , the membrane p o t e n t i a l ( V ) was kept c o n s t a n t a t t h e r e s t i n g l e v e l by D C - c u r r e n t i n j e c t i o n . In both c e l l s V was d e p o l a r i z e d by 13 and 15 mV r e s p e c t i v e l y a f t e r ~25 m i n . (C) complete s u p p r e s s i o n o f an IPSP a f t e r b i c u c u l l i n e a p p l i c a t i o n (BIC; 50 uM; 5 min) i n a neuron impaled by a CS2SO4 e l e c t r o d e . (D) time course of blockade of the IPSPs in a sensorimotor neuron p e n e t r a t e d w i t h a CS2SO4 m i c r o p i p e t t e . Lower and m i d d l e t r a c e s were o b t a i n e d a f t e r 1 and 3 min of b i c u c u l l i n e (50 uM) perfusion. In (C) and ( D ) . IPSPs were evoked a f t e r complete a t t e n u a t i o n of t h e AHP and a t t a i n m e n t o f a steady s t a t e o f V (-48 mV from a r e s t i n g l e v e l -66 mV). +  +  m  m  m  H. EL-BEHEIRY  removed any " c o n t a m i n a t i o n " o f t h e IPSPs by K-mediated potentials.  In  addition,  a m p l i t u d e o f t h e evoked examination  0.013  (±0.007)  hyperpolarizing  depolarization  increased  IPSPs and rendered them more amenable  of drug-actions.  whereas  (±0.35)  the Cs-induced  late  The mean  amplitude  mV/ms r e s p e c t i v e l y ( c f . Howe e t a l .  were  410  was 5 . 2 mV ms and  (^42)  Sutor  1987;  the  f o r extensive  o f t h e IPSPs  t h e mean d u r a t i o n and r a t e o f decay  61  and H a b l i t z  1989).  The IPSPs and  application  of  i n a l l neurons  reproducible  prolonged usually IPSPs  on r e p e a t e d  both  agents.  after  than  E C  Although  values  2 0 min. 5  Q  The E C ^ Q  f o r depression  a dose-dependent  than t h a t f o r t h e EPSPs. control  t o t h e same  were  min) i n t h e case  (12-15  e v i d e n t , t h e dose-response  the  applications  depressed the  was d o s e - d e p e n d e n t ,  1 5 and 1 6 ) . The e f f e c t s  administration  was observed  effect  consistently  reversible  neuron.  Isoflurane  ( 7 5 uM) depressed t h e a m p l i t u d e s o f IPSPs by ~ 5 4 and  (Figs.  was g r e a t e r  or A l t h e s i n  (n = 1 7 ) ; t h i s  ( 1 . 5 MAC) and A l t h e s i n 55% r e s p e c t i v e l y  isoflurane  curves  o f both  evident  agents.  o f t h e EPSPs  slowing  of  f o r both agents  after  Recovery  f o r the attenuation  of the  i n t h e case  t h e IPSP  had a l o w e r  The maximum d e p r e s s i o n s  were  only  decay was  slope  values  o f t h e r a t e o f decay  ~ 5 4 % f o r 2 . 5 MAC i s o f l u r a n e  of  from  and 4 5 % f o r 1 0 0 uM  Althesin.  S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d i n t h e h i g h e r  dose range  (Fig.1 6 ) .  Moreover,  complete r e c o v e r y  o f t h e r a t e o f decay was  observed i n ~ 7 0 % o f neurons. Discussion  4.2 The  early  studies  L a r r a b e e and Posternak mission  was reduced  by E c c l e s (1952)  by  (1946),  demonstrated  administration  Brookes that of  and E c c l e s  excitatory  various  i n c l u d i n g c h l o r o f o r m , d i e t h y l e t h e r and b a r b i t u r a t e s .  (1947)  synaptic  general  and  trans-  anaesthetics  L a t e r s t u d i e s by Somjen  control  IFL 1.5 MAC  recovery  250ms  B  controls  recoveries  AL 35,,M  5mV  AL 75/iM  F i g . 15 IPSP-attenuation by i s o f l u r a n e and A l t h e s i n a f t e r K - c o n d u c t a n c e b l o c k a d e by i n t r a c e l l u l a r a p p l i c a t i o n of C s . (A) i s o f l u r a n e (1.5 MAC; 12 min a p p l i c a t i o n ) suppressed t h e IPSPs by ~60% . (B) A l t h e s i n (35 and 75 L.M; 12 min a p p l i c a t i o n ) a t t e n u a t e d of t h e IPSPs evoked i n s e n s o r i m o t o r and a n t e r i o r c i n g u l a t e neurons r e s p e c t i v e l y . In (A) and (B) t h e superimposed t r a c e s were at l e a s t 6 i n each c o n d i t i o n . +  +  H. EL-BEHEIRY  63  B AL^5/iM  IFL1MAC \  C 100 -r  OAmplitude ARate of decay  O Amplitude A Rate of decaj BO - -  o *W  80 +  o  a*  &  « 20--  0.5  1.0  -I > 10.0 20.0  2.0  Isoflurane (MAC)  ' 50.0  r— 100.0  Althesin (JJM)  F i g . 16 Dose-dependent i n t e r f e r e n c e with the IPSPs i n neocortical neurons with concomitant blockade of K -conductances by i n t r a c e l l u l a r Cs . (A and B) computer-averaged traces i n two neurons i n which i s o f l u r a n e and A l t h e s i n (IFL, 1 MAC; AL, 75 uM) were a p p l i e d . (C and D) dose-response curves f o r i s o f l u r a n e and A l t h e s i n showing suppression of the amplitudes and rates of decay by at l e a s t 3 anaesthetic a p p l i c a t i o n s f o r each dose. +  +  H. EL-BEHEIRY and G i l l  (1963) and Somjen  a t e s depressed for  direct  Subsequently, synaptic  depress  anaesthetics  halothane  levels,  and in  Miu  and  e t a l . , 1975).  the  Puil  However,  central were  of  Langmoen and  has  particularly  1986a,b  and  (1972) has  now been in  the  the d e p r e s s i v e  his  associates  actions  (1988)  of  also  e n f l u r a n e on evoked EPSPs clear  evidence  increases expected  the to  at  found in  present  e f f i c a c y of  prevail  general no  the  that  excitatory the  t r a t i o n (see b e l o w ;  any  in vitro.  halothane  or  There  of  the  during  the  preparations.  is  no  actually might  be  anaesthetic  anaesthetic  could  adminis-  anaesthetics  on i n h i b i t o r y  synaptic  transmission  E c c l e s and  i n h i b i t i o n was enhanced  by  associates  barbiturate  a d m i n i s t r a t i o n but d e p r e s s e d by d i e t h y l e t h e r and c h l o r a l  application. increased  and  1978).  reported that presynaptic  chloralose  Fujiwara  inputs to i n h i b i t o r y interneurons,  a r e more complex t h a n on t h o s e a t e x c i t a t o r y synapses. (1963)  resistant  anaesthetic  phase  Roth  dendro-  Such a mechanism  excitatory  to  Richards  the unusual  isoflurane,  general  found  1973;  Moreover,  slice  i n h i b i t o r y processes  c f . Morris  The a c t i o n s o f g e n e r a l  of  synapses.  early  state or, given e x c i t a t o r y synaptic c o n t r i b u t e t o t h e enhanced  effects  hippocampal  time,  during  anaesthetics.  the  and  d e n d r i t i c e x c i t a t o r y synapses o f t h e o l f a c t o r y bulb a r e r e l a t i v e l y to  At  hippocampal  Maclver  (Richards  shown t h a t  excitatory  1969).  1987;  cortex  obtained  transmission.  depress  (Galindo  anaesthetics  olfactory  Nicoll  synaptic  shown t o  nucleus  transmission  and  1989)  on  cuneate  variety  synaptic  (Berg-Johnson  Hence c l e a r e v i d e n c e was  barbiturates  a wide  excitatory  formation 1988;  of  transmission  supraspinal  (1963) c o n f i r m e d t h a t d i e t h y l e t h e r and b a r b i t u r -  the EPSPs i n motoneurons.  actions  64  or  hydrate  L a r s o n and M a j o r (1970) r e p o r t e d t h a t h e x o b a r b i t o n e a p p l i c a t i o n duration Later,  of Nicoll  IPSPs and  in his  the  spinal  associates  cord (1975)  in  in vivo also  feline  observed  a  H. EL-BEHEIRY  65  barbiturate-induced prolongation  of i n h i b i t o r y p o t e n t i a l s  in the  hippocampus  of i n v i v o f e l i n e p r e p a r a t i o n s .  More r e c e n t l y , i n c r e a s e s  i n the  spontaneous  IPSCs  (Gage and R o b e r t s o n  have been observed studies  and  i n hippocampal  the  by  alphaxalone  Nicoll  (1972),  and ketamine  the s p i n a l  in  (Takahashi  in  the  attenuated 1985;  by  CA1  1987).  clinically  used  vulnerable  extensively  procedures from o t h e r  cells  of  of  In  1980)  1989)  in vitro  which  were  postsynaptic  inhibition.  any  the  the  inhalational  specific  depression  newborn  evoked  increase of  rat  spinal  are  anaesthetics  in  IPSPs was  inhibitory  hippocampus  to  transients  during  neocortex  which  the  actions  (cf.  i s o l a t e the  "contaminating"  investigations.  contradictions previous  in  to anaesthetic  the previous  cord  potentials  consistently  (Yoshimura  arising  investigators  4.2.1 anaesthetic amplitudes  Anaesthetic effects with  isoflurane  5 0  is  an  Introduction).  associated  conductances  have  et a l .  not  been  that  very  is  addition, with the  been  EPSPs 1 MAC  is  EPSPs  or  implemented  in  apparent  preparations  some d i f f i c u l t y  of  EPSPs.  the  dose-dependent  and  these  experi-  used by in  inter-  transients.  depression  and A l t h e s i n ;  have not  T h i s may have r e s u l t e d i n some o f t h e  and a l s o may have p r o v i d e d  of  area In  from the d i f f e r e n t e x p e r i m e n t a l  on  EC s  anaesthesia  conductances  p r e t i n g the " t r u e e f f e c t s " on t h e s e  by  Puil  F u j i w a r a e t a l . 1988).  investigated  IPSPs  and  halothane,  of  Synaptically  M o d i f i c a t i o n of the s y n a p t i c  mental  1978,  i n v i v o whereas  preparations  pyramidal  (Miu  barbiturates,  (1968) d i d not observe  slice  and T a k e n o s h i t a  recorded  of  increased the duration  c o r d , Weakly  in vitro  IPSPs  (Sc hoi f i e l d  administration  t h e d i r e c t i n h i b i t i o n o f motoneurons evident  evoked  in vitro s l i c e preparations.  of t h e o l f a c t o r y bulb and c o r t e x  initiated  In  1985)  A  ~50 LIM f o r  concentrations  striking  the are  feature  depression respective similar  of in  the their  depressions  t o those  that  H. EL-BEHEIRY  have  been  1974;  in  investigations  the  synaptic (1975  aliphatic  alcohols  junction. neurons EPSP  actions.  in  1986b;  its  have  Zorychta  or  neocortex  could  excitability Saint  1988;  Kullman  conductance  (Nicol  sensitivity  and  Linder  in the  the  due  to  and  presynaptic  Saint  and  certain  neuromuscular  or  1986;  Butterworth et a l  inhibiting quantal  the  content  and A n i s  1984;  1982)  (Anis  or  1989)  and, of  or and  depression transmitter  of  released  quanta  (Cheng  and  Brunner  1978;  an  1985;  Bosnjak  increase  decrease  e t a l . 1983;  of  number  Johnson  a  in  postsynaptic  Berg-Johnsen  synthesis  Postsynaptically,  Madison  to a t r a n s m i t t e r  the  decay  Gage of  al the  The a n a e s t h e t i c d e p r e s s i o n  i n t e r f e r i n g with  1989).  and  and  effects at  in  r a t e of  (1975)  similar  1989).  (Quastel  Lodge  al  et  i n c l u d e d e p r e s s i o n o f impulse c o n d u c t i o n  e i t h e r by  1978; et  (Smith  have not been p r e v i o u s l y observed  be  e t a l . 1986;  decreasing  and Capek  slowing  reported  Kullman e t a l  m o b i l i z a t i o n , thereby  impulse  animals  on the m i n i a t u r e e n d p l a t e c u r r e n t s  of t r a n s m i t t e r release  al.  Quastel  P r e s y n a p t i c mechanisms  terminal  and/or  t h e dose-dependent  1979)  of the CNS ( c f .  Langmoen  anaesthetized  An i n t e r e s t i n g e f f e c t w h i c h was observed  was  and  of  However, comparable f i n d i n g s  amplitude  nerve  brain  transients.  associates  per  the  M i l l s e t a l . 1987).  present of  determined  66  in  Sawada  in  et  membrane  the  receptor  and Yamamoto  1985)  can l e a d t o a t t e n u a t i o n o f EPSPs. The observed s u p p r e s s i o n summation  of  agents.  The  "isolated" that  p r e - and  of the EPSP a m p l i t u d e s c o u l d be e x p l a i n e d by a  postsynaptic  depression  of  the  i n n e o c o r t i c a l neurons  a Ca-conductance  may  be  depressant  actions  Ca-activated  of  K-conductance  ( c f . S e c t i o n 6) p r o v i d e s  depressed  in  the  the  anaesthetic that  was  i n d i r e c t evidence  presynaptic  terminal.  For  2+ example,  an  attenuated  r e l e a s e d per s t i m u l u s .  Ca  influx  would  This p o s s i b i l i t y i s  decrease  the  number  of  quanta  s u p p o r t e d by t h e o b s e r v a t i o n s  of  H. EL-BEHEIRY  Krnjevic  and  dependent  Puil  Ca  (1988)  currents  who  in  number  depression  of  of  hippocampal  c f . Morgan and B r y a n t  Z o r y c h t a and c o l l a b o r a t o r s  t h a t the decreased  a  voltage-clamped  halothane a p p l i c a t i o n ( a l s o 1983).  observed  certain  CA1  1977  voltage-  neurons  and N i s h i  released during  e t h e r a n a e s t h e s i a was the mechanism of  suppression  during  and Oyama  (1975; Z o r y c h t a and Capek 1978)  quanta  67  reported  halothane and  diethyl-  of monosynaptic  EPSPs  in  t h e s p i n a l c o r d of c a t s . The  prolongation  of  the  decay  with  a  concomitant  depression  of  the  a m p l i t u d e of t h e EPSPs c o u l d be due t o : (a)  a proportional  M i n c h i n 1981) (b)  supression  of the uptake mechanisms  of  the membrane  i n p u t c a p a c i t a n c e a t membrane  sites  e l e c t r o d e ( c f . P u i l and Gimbarzevsky an i n c r e a s e  mediating  Puil,  1981;  w i t h a r e d u c t i o n i n t h e number of r e c e p t o r s a c t i v a t e d ;  a prolongation  (c)  (cf.  time c o n s t a n t  due  d i s t a n t from t h e  to  an  increased  somatic  recording  1987);  i n t h e mean open t i m e of t h e r e c e p t o r - o p e r a t e d c h a n n e l s  the EPSPs, c o n c o m i t a n t w i t h a decrease  i n the a b s o l u t e  number  of r e c e p t o r s a c t i v a t e d or d)  the  simultaneous  depression  of  the  IPSPs  during  the  anaesthetic  The r e s u l t s of t h e p r e s e n t s t u d i e s are a t v a r i a n c e w i t h t h e  observations  application.  reported  in olfactory  attributed  the  cortex  depression  of  and  hippocampal  the  EPSPs  in  formation.  olfactory  Scholfield  cortex  e f f e c t produced by t h e a n a e s t h e t i c p r o l o n g a t i o n o f t h e IPSPs. investigations anaesthetic  or  a  shunting  In t h e p r e s e n t  b i c u c u l l i n e (50 uM) was a p p l i e d t o n e o c o r t i c a l neurons  a p p l i c a t i o n and t h e  to eliminate t h i s p o s s i b i l i t y . ane  to  (1980)  Althesin  was  perfused  IPSPs were a d d i t i o n a l l y m o n i t o r e d  in  Once b l o c k a d e o f IPSP was o b s e r v e d , simultaneously  with  bicuculline.  before order  isoflur-  Clinically  H. EL-BEHEIRY  relevant  doses  effective  in  of  both  inhibiting  the GABA^-antagonist the  CA1  neurons  enflurane According  agents  or  the  (cf.  were  not  only  not  In t h e  affected  (Yoshimura  t o the l a t t e r  the  e p i l e p t i f o r m neuronal  F i g . 12).  halothane  depressed  reports,  by  EPSPs  discharges  hippocampus,  the  et a l .  but  EPSPs  applications 1985;  a non-air-tight  gas  also  by  recorded  in  isoflurane,  et a l .  chamber  was  t h e a n a e s t h e t i c s were a p p l i e d o n l y by bath p e r f u s i o n and were not i n t o the atmosphere  above the s l i c e .  not bubbled i n t o the p e r f u s a t e added  and  "shaken  application  have o c c u r r e d d u r i n g the  concentrations  1988).  used  and  introduced  a d d i t i o n , the v o l a t i l e a g e n t s  were  f o r a s u i t a b l e e q u i l i b r a t i o n p e r i o d but were  vigorously"  i n the chamber.  In  were  initiated  of  Fujiwara  68  in  a  stock  solution  T h e r e f o r e , leakage  before  dilution  of a n a e s t h e t i c  vapour  and could  such a p p l i c a t i o n s and c o u l d have l e a d t o a d e c r e a s e of  the  agents  at the  effector  sites  (cf.  Bazil  et  in al.  1987). 4.2.2  Anaesthetic  actions  on  the  IPSPs.  e l e c t r o d e s i n t h e s e e x p e r i m e n t s p r o v i d e d two main (a)  lower t i p  resistances  than  in  the case  The  use  of  CsgSO^  advantages: of  K-acetate  filled  micro-  p i p e t t e s and (b) b l o c k a d e o f K - c o n d u c t a n c e s +  t h a t c o u l d have c o n t a m i n a t e d endogenous  a c t i v i t y mediated by GABA evoked C l " i n f l u x . The dose-dependent decrease  in  mechanism  for  inhibitory  the  rate  depression of  decay  the d e p r e s s i o n  interneurons  are  of  of  the  IPSP  suggests the  IPSPs  activated  a and by  amplitudes common the  together  p r e - and EPSPs.  excitatory  with  the  postsynaptic  Nonetheless, synapses,  if the  a n a e s t h e t i c - i n d u c e d d e p r e s s i o n o f EPSPs may l e a d t o a c o n c o m i t a n t a t t e n u a t i o n of t h e IPSPs.  H. EL-BEHEIRY Takahashi  and  Takenoshita  (1987)  also  observed  evoked EPSPs and IPSPs r e c o r d e d from motoneurons tions.  However,  their  results  and  those  of  an  attenuation  the  present  and IPSPs r e p o r t e d f o r o l f a c t o r y c o r t e x and hippocampal Gage  and  Robertson  fundamental  differences  neocortical  (Matsumura  olfactory have  cortical  arisen  Harrison  between t h e et  al.  neurons  from t h e  1985;  types  1988),  +  ( S c h o l f i e l d 1978),  a c t i v a t i o n of  had not been a p p l i e d .  (20-25°C) cortex ionic  used  during  channels  cortex,  an  and  t h e enhancement  of t h e  increase  1987).  in  is  the  slow mean  that  the  also  the f u l l  1984)  (Smith  et a l .  1974).  (i.e.,  In  was  At  a  of  activated  the  olfactory Hence,  suppresion  i n t h e b r a i n grey m a t t e r o f  such  low c o n c e n t r a t i o n s  not  alphaxalone  induce have  anaesthesia,  ionic  of  EPSPs  currents. neurons  neurons o f v e r t e b r a t e s ( C u l l e n and M a r t i n  of  since  GABAergic  1982).  anaesthetized  the  by p r o l o n g i n g  particularly  revealed a depression  in  the  olfactory  observed.  e x t e n t of the i n h i b i t o r y  a l p h a x a l o n e may a c t as a s e d a t i v e , e . g . , likely  could  t h e doses w h i c h were used ( 0 . 1 - 1 0 uM) were s i x  lower t h a n t h o s e t h a t o c c u r  and  where i n t e r n a l  In t h e case o f t h e a l p h a x a l o n e a p p l i c a t i o n s to c u l t u r e d hippocampal ( H a r r i s o n e t a l . 1987),  the  the low t e m p e r a t u r e  open-time.  the EPSPs  in  findings  kinetics  IPSPs c o u l d be a t t r i b u t e d t o  w h i c h c o u l d have unmasked  inputs  the  "contaminate"  allocortex  down  (Scholfield  (Buzsaki  that  IPSCs  Despite  and hippocampal neurons  to  of  neurons  CA1  +  their  depression  and evoked  synaptic  K -conductances  experiments  the  investigations  the d i s c r e p a n t  possibility  would t e n d  may  anaesthetic  Another  the  and hippocampus)  tonic  hippocampal  IPSPs i . e . , i n the o l f a c t o r y c o r t i c a l Cs  et a l .  of  of  in i n v i t r o s l i c e prepara-  c o n t r a d i c t the a n a e s t h e t i c p o t e n t i a t i o n of the spontaneous  1980;  69  the  i n vivo  times rats  situation  I P S P s , but would  previous responses  studies in  with  midbrain  H. EL-BEHEIRY  5  70  ANAESTHETIC INDUCED ALTERATIONS IN NEURONAL RESPONSIVENESS TO ACTIVATING TRANSMITTERS AND RELATED SUBSTANCES  The mean (±SD) included in t h i s These neurons response  to  resting  p o t e n t i a l and i n p u t r e s i s t a n c e  s e r i e s of experiments were -70  f i r e d spikes  f o r the 65  (^2.25) mV and 51 (^2.5) M&.  o f >70 mV a m p l i t u d e and were <2 ms i n d u r a t i o n  depolarizing  current  pulses.  The  five-barrelled  inserted  separately  at  the  surface  of  the  slice  (cf.  r e c o r d i n g m i c r o e l e c t r o d e s were f i l l e d w i t h KCl (3 M) o r K S 0 2  5.1  (0.6  The  M).  Results 5.1.1  Control responses to i o n t o p h o r e t i c a l l y a p p l i e d 5.1.1.1  Acetylcholine.  (*SEM) d e p o l a r i z a t i o n  were  l o c a t e d i n an a r e a c o r r e s p o n d i n g  However,  at more s u p e r f i c i a l  long-lasting The using  input  resistances  due  in  to  depolarizations  of  the  slow  signs of d e s e n s i t i z a t i o n  largest  responses  and I I I ) ,  pulses  input  (delayed) as  ( F i g . 17A1). (20 uM;  Wong and  and were u s u a l l y  the  by  to  the  These  in  actions  t o ACh a p p l i c a t i o n u s u a l l y  injected with depolarizing current pulses.  decay  same  3 neurons).  the  that  induced a  Gallagher  1989).  by  decreased, ACh-induced  ( F i g . 17)  neuron,  tests  (~30%)  were The  a  neocortex.  increased  resistances  as  neurons  repetitive  rectification. well  50  evoked  ACh a p p l i c a t i o n  monitored  were  of  and V of  neurons  application  a p p l i c a t i o n of scopolamine  IV  agents  (50-350 nA)  out  (cf.  in onset  repeated  36  to layers  (II  ACh  i n 3 neurons  neurons  membrane  were  on  4  of  (-±1.5) mV i n  layers  hyperpolarizing  applications;  reproducible  12.3  hyperpolarization  constant  probably  of  Applications  mean  such  pipettes  Methods). 4  in  iontophoretic  e l e c t r o d e s w h i c h were used h e r e , were e i t h e r g l u e d t o t h e r e c o r d i n g or  neurons  without  and  were  obvious  c o u l d be b l o c k e d by t h e  Neurons t h a t e x h i b i t e d the  fired  spikes  in doublets  These c e l l s had i n p u t  when  resistance  A1  A2  ACh  ,  ,  —1  H. EL-BEHEIRY  71  I  20 s Fig. 17 Effects of iontophoretically applied acetylcholine (ACh) on n e o c o r t i c a l neurons. (A) a t y p i c a l l y slow onset response t o ACh e j e c t e d from a compound e l e c t r o d e assembly i s shown f o r a s e n s o r i m o t o r neuron. (A2) In the same neuron i n j e c t i o n s of j u s t - t h r e s h o l d d e p o l a r i z i n g c u r r e n t p u l s e s evoked a c t i o n p o t e n t i a l s i n d o u b l e t s . Note t h e l a r g e amount of spontaneous s y n a p t i c a c t i v i t y as w e l l as the sagging h y p e r p o l a r i z i n g response t o c u r r e n t pulse i n j e c t i o n . (B) response of a s e n s o r i m o t o r neuron t o ACh a p p l i e d from a s e p a r a t e e l e c t r o d e c o n s i s t e d of a s m a l l d e p o l a r i z a t i o n and increased s p i k i n g a c t i v i t y which was preceded by a b r i e f h y p e r p o l a r i z a t i o n . (CI) ACh evoked a d e p o l a r i z a t i o n w i t h an a f t e r b u r s t of s y n a p t i c a c t i v i t y d e s p i t e the p r e s e n c e o f TTX. H y p e r p o l a r i z i n g v o l t a g e responses t o i n t r a c e l l u l a r c u r r e n t injections before, during, and a f t e r ACh a p p l i c a t i o n are shown i n ( C 2 ) .  H. EL-BEHEIRY  o f a p p r o x i m a t e l y 50 Mn, and e x h i b i t e d a l a r g e amount o f spontaneous activity  as  injections  well  as  'sagging  ( F i g . 17A2).  responses'  to  hyperpolarizing  72  synaptic  current  The responses t o ACh c o u l d be grouped  pulse  i n t o two main  categories: (1)  depolarizing  ( F i g . 17C1)  that  response was  with  observed  an  on  afterburst  several  of  synaptic  occasions  in  the  activity  presence  of  TTX and (2)  depolarizing  ( F i g . 17B);  response  this  iontophoretic  that  was  hyperpolarizing  e l e c t r o d e was  preceded  effects  of ACh,  offset,  i.e.,  neurons  Glutamate  independent  the were  and  responses an  from  associated  t o Glu were a b r u p t type  of  The mean (±SEM)  In  brief of  cf.  response  period  the  depolarizing  uncommon 0.7-1,  (2-3 s)  to  for  the  equal  of  (Figs.  ratio unity  and  Puil  (cf.  of  Glu-evoked  pulses  1972).  of  "ACh/Glu  (Fig.  18C).  depolarizations  18A-C).  rapid  All  ~40-80% ( F i g . 18A2).  In  and  produced  ~15% o f  was  1983). which  a tendency  had i n t e r t i p by  the  65  to Glu  at It  the was  varied  towards  distances  consistently  spaced  a end not  between a  higher  o f 50 nm. ejection  of e q u i v a l e n t c u r r e n t were remarkably r e p r o d u c i b l e d e s p i t e t h e  of the experimental p e r i o d ( e . g . , 3 h r . ) .  Bath  neurons,  observed  Smith  depolarizations" There  of t h e neurons  (2-4 mV) was  Padjen  r a t i o when the e l e c t r o d e i n t h e a s s e m b l i e s The  had a  the  (40-300 nA) and d i d not e x h i b i t  resistance  hyperpolarization  response  and  to  d e p o l a r i z a t i o n was 20.25 ( ± 1 . 4 ) mV and was  input  ZieglgSnsberger  the  electrode,  contrast  i n onset  a p p l i c a t i o n o f TTX had no e f f e c t on the r e s p o n s i v e n e s s ( F i g . 18B;  o b t a i n e d when  recording  N-methyl-D-aspartate.  "on/off"  w i t h a decreased  the  hyperpolarization  nM TTX.  were d e p o l a r i z e d by Glu a p p l i c a t i o n s  desensitization.  a  p o t e n t i a l which was  c o u l d be b l o c k e d w i t h a p p l i c a t i o n of 1-1.5 5.1.1.2  by  length  H. EL-BEHEIRY  Glu  73  ACh  F i g . 18 Glutamate ( G l u ) evoked responses i n n e o c o r t i c a l neurons. ( A l ) Glu a p p l i c a t i o n induced a f a s t " o n / o f f " t y p e o f d e p o l a r i z i n g response w h i c h was a s s o c i a t e d w i t h an i n c r e a s e d conductance (A2). (B) t h e Glu a c t i o n s were not a f f e c t e d by t h e p r e f u s i o n of TTX ( 1 . 5 uM). (C) a l t e r n a t i n g a p p l i c a t i o n s o f Glu and ACh on an a n t e r i o r c i n g u l a t e neuron produced d e p o l a r i z i n g r e s p o n s e s (ACh/Glu r a t i o = ~0.85) w h i c h are shown t y p i c a l l y h e r e .  H. EL-BEHEIRY  Similarly  b r i e f NMDA a p p l i c a t i o n s  depolarized  15/37  neurons  74  despite  the  +2 presence  of  2 mM  Mg  in  the  d e p o l a r i z i n g responses t h a t a l s o was  apparent  after  the  in conjunction  peak  of  responses a l s o were (1)  the  bath.  NMDA  had a slow decay  w i t h an  potential  increased  response  input  d e p o l a r i z i n g response  a relatively  5.1.1.3  fast  Two  response  that  other  t h a t e x h i b i t e d an c u t e form o f  waned  types  of  (Fig.  19C;  4  hyperpolarization  which  desensitization  5 neurons) and  G l u - l i k e response  that  desensitized  markedly  on  neurons).  Gamma-aminobutyrate  electrode assemblies (2)  this  resistance  and  baclofen.  a p p l i c a t i o n o f GABA produced two t y p e s of responses (36 a  developing  observed:  repeated a p p l i c a t i o n s  (1)  slowly  ( F i g . 19A);  ( F i g . 19A).  d u r i n g t h e a p p l i c a t i o n p e r i o d ( F i g . 19B; (2)  induced  was  observed  Iontophoretic  neurons):  only  when  the  compound  were used (9/9 neurons) and  a d e p o l a r i z a t i o n w h i c h was evoked when t h e a p p l i c a t i o n p i p e t t e was  s e p a r a t e from t h e r e c o r d i n g e l e c t r o d e (29/29  neurons).  The hyperpol a r i z i n g r e s p o n s e s (5-9 mV) t o GABA were a s s o c i a t e d w i t h decreased input resistance as w e l l  during  (40-60%).  An acute d e s e n s i t i z a t i o n  the a p p l i c a t i o n p e r i o d .  Moreover,  i z a t i o n was e v i d e n t when t h e d u r a t i o n  ( F i g . 20)  a long-lasting  of GABA a p p l i c a t i o n was  was  observed  hyperpolarincreased  to  >10 s ( c f . B l a x t e r e t a l . 1986). The increase  depolarizing in  input  type  of  conductance  response and  (14-40 mV)  could  be  further  was  accompanied  by  an  subdivided  into  three  (2) w i t h a c u t e d e s e n s i t i z a t i o n d u r i n g t h e a p p l i c a t i o n p e r i o d  (Fig.  21B),  forms: (1) w i t h o u t d e s e n s i t i z a t i o n ( F i g .  and  21A),  H. EL-BEHEIRY  NMDA  75  Glu  F i g . 19 NMDA-induced d e p o l a r i z a t i o n s i n s e n s o r i m o t o r l a y e r V neurons. (A) NMDA a p p l i c a t i o n evoked slow d e p o l a r i z i n g response w i t h i n i t i a l i n c r e a s e i n input r e s i s t a n c e (lower t r a c e s ) . (B) a c u t e d e s e n s i t i z a t i o n e x h i b i t e d by a n o t h e r neuron i n response t o NMDA e j e c t i o n from a s e p a r a t e p i p e t t e . (C) a t h i r d type o f NMDA a c t i o n was a f a s t G l u - l i k e r e s p o n s e . This response d e s e n s i t i z e d markedly on r e p e a t e d a p p l i c a t i o n .  GABA  NaCl  GABA  F i g . 20 A h y p e r p o l a r i z i n g response t o GABA a p p l i e d from a compound e l e c t r o d e assembly. (A) a c u t e d e s e n s i t i z a t i o n o f t h e GABA-evoked response i s shown on the l e f t and l o n g - l a s t i n g hyperp o l a r i z a t i o n on the r i g h t . (B) The h y p e r p o l a r i z i n g response i n another neuron was b l o c k e d by b i c u c u l l i n e a f t e r 5 min and r e c o v e r y was o b t a i n e d a f t e r 20 min a t a f l o w r a t e of 2 m l / m i n .  H. EL-BEHEIRY  77  GABA  F i g . 21 Different types of depolarizing application of GABA. Non-desensitizing (A) responses are shown for two sensorimotor before, at the peak, and a f t e r a depolarizing (C). TTX.(1.5 uM) was applied in (A and B ) .  responses evoked by dendritic and acutely desensitizing (B) neurons. Conductance changes e f f e c t of GABA are evident in  H. EL-BEHEIRY  (3)  with tachyphylaxis  several  occasions  with the r i s i n g  on f r e q u e n t r e p e a t e d a p p l i c a t i o n s  the  GABA d e p o l a r i z a t i o n s  and/or  f a l l i n g portions  were  (Fig.  associated  of each response  78  22C).  with  On  spikes  (Figs.  21C  and  22C). In o r d e r t o d e t e r m i n e i f the d e p o l a r i z i n g i.e.,  decreased  induce  spike  application  membrane  activity of GABA  excitability,  and  (Fig.  this  was  22B).  response was indeed  Glu  was  followed  by  The d e p o l a r i z i n g  applied a  continuously  concomitant  response  s u p p r e s s e d the a c t i o n p o t e n t i a l s  evoked by G l u a p p l i c a t i o n .  the  was  inhibition  of  spike  firing  not  likely  inhibitory,  attributable  d e p o l a r i z a t i o n because t h e combined d e p o l a r i z a t i o n was j u s t  pulse-like  induced In  TTX  to  22A).  i n j e c t i o n t o l e v e l s between -100  determine The  a  reversal  depolarizations  hyperpolarizing  values  of  potential  evoked the  by  to  the  GABA  4 min  hyperpolarizing GABA-evoked  of  bicuculline  GABA i n c r e a s e d membrane  response  was  depolarization  (50 uM)  blocked  in  suprathreshold.  was  blocked  potential.  GABA.  Baclofen  baclofen  when a p p l i e d  iontophoretically  Iontophoretically  conductance  hyperpolarized and  did  (Fig.  23B).  the The  F i g . 23A).  not  of  A concomitant  d i d not a l t e r t h e response from a  3  separate  neurons  affect  pipette  (6 n e u r o n s ) .  Glu  by  to did  However,  2-3 mV  with  depolarizations.  a p p l i e d b a c l o f e n d i d not depress the number,  shapes of G l u - e v o k e d s p i k e s  neurons)  application  baclofen  in  (3  prolonged  (50 uM)  an  22A).  by  only  bath  10-15% i n c r e a s e  at  F i g . 20).  p o t e n t i a l o r i n p u t conductance  of  (Fig.  more  2 ml/min;  not change t h e r e s t i n g application  at  However,  of  (Fig.  rate  b i c u c u l l i n e i n doses as h i g h as 150 uM (5 neurons; e x t r a c e l l u l a r a p p l i c a t i o n of  displaced  amplitude  application  (flow  excessive  depolarization  imposed p o t e n t i a l of ~-30 mV the response was m u l t i p h a s i c After  cases,  and -30 mV i n t h e presence  for  resting  by GABA  such  GABA was a p p l i e d t o 4 neurons w i t h r e s t i n g p o t e n t i a l s t h a t were by D C - c u r r e n t  to  amplitudes  or  H. EL-BEHEIRY  79  15 mV  10 mV  GABA  GABA  5s 15 mV  F i g . 22 C h a r a c t e r i s t i c s o f t h e GABA-evoked d e p o l a r i z a t i o n s i n n e o c o r t i c a l neurons. (A) s h i f t i n g the r e s t i n g membrane p o t e n t i a l by i n t r a c e l l u l a r D C - i n j e c t i o n r e v e a l e d a b i p h a s i c response beginning a t V <-40 mV. (B) c o n c o m i t a n t a p p l i c a t i o n o f GABA shunted t h e s p i k e s evoked by G l u . (C) r e p e a t e d a p p l i c a t i o n of GABA of <10 s i n t e r v a l s d e s e n s i t i z e d the neuron c o m p l e t e l y t o t h e subsequent GABA a p p l i c a t i o n s . The s p i k e s on top o f t h e co-ejected GABA r e s p o n s e s were p r o b a b l y due t o e x c i t a t o r y a c t i o n s o f H w i t h the a g o n i s t . m  +  H. EL-BEHEIRY  80  F i g . 2 3 B i c u c u l l i n e , i n high doses, blocked the GABA-induced d e p o l a r i z a t i o n s i n n e o c o r t i c a l neurons. ( A ) b i c u c u l l i n e (150 M b l o c k e d the d e p o l a r i z i n g r e s p o n s e s t o d e n d r i t i c a p p l i c a t i o n o f G A B A whereas t h e G l u - r e s p o n s e s were not a f f e c t e d . (B) b a c l o f e n , a G A B A B a g o n i s t , had no e f f e c t on V o r on the Glu-evoked spikes i n a sensorimotor neuron.  H. EL-BEHEIRY  5.1.2  Anaesthetic-induced  alterations  inhibitory  transmitter substances.  excitatory  or  inhibitory  excitatory  and  The observed changes i n r e s p o n s e s t o  the  transmitter  in  responses  substances  which  to  81  are  described  below  were not a s s o c i a t e d w i t h marked changes i n p a s s i v e membrane p r o p e r t i e s .  For  example,  and  200 uM  only  the  highest  respectively)  applications  effects  of  isoflurane The  and  in  25-200 uM)  and  neurons  were  of  perfusion.  depressed  (Figs.  Althesin  suppression  the  full  recovery  24  Althesin  by  anaesthetics  dose-dependent  and  was  higher  than  depressed  reversible  isoflurane-induced  attenuation  that  for  the  the  manner of  ACh  the  ACh  and 2 5 ) .  In  contrast,  the  by  50% depressed  40%. were  response  was  high  the  Glu-induced ( F i g . 26  the  Glu-responses  Althesin the  and  27A). was  concentration  affected in  10  neurons,  by  either  agent.  after  recovery  was  recovery  application Fig.  The  EC  a the  which  is  ACh-induced  which  that  in for  5 Q  ~1.9 MAC of  of  25B).  suppressed by  about  ( F i g . 28) in  i s o f l u r a n e a p p l i c a t i o n enhanced t h e Glu responses and almost t o t a l l y ACh a c t i o n s .  the  r  evident  depolarizations  Note  a  75 nM  depolarizations  depression  Glu-evoked  f°  partial  dose  in  and  full  Usually,  on  Althesin  responses  0.9 MAC  on t h e dose,  observed  and  The G l u - i n d u c e d r e s p o n s e s were p o t e n t i a t e d i n 4 neurons not  decreased  The ECf^s  responses  ml/min).  corresponding  depolarizations. ACh  and  Isoflurane  e i t h e r agent ( 2 - 2 . 5 MAC i s o f l u r a n e and 150-200 uM A l t h e s i n ; Both  ( 2 . 5 MAC  3-5 mV  approximately  Depending  a f t e r 8-16 min ( f l o w r a t e 1.5-3 some c a s e s ,  and  G l u and NMDA.  r e v e r s i b l e manner  6-12 min of a n a e s t h e t i c observed  the  Responses t o ACh,  and  respectively.  isoflurane  by <20% .  ( 0 . 5 - 2 . 5 MAC;  dose-dependent  of  hyperpolarized  t h e i r input resistances 5.1.2.1  doses  and  Fig.'28, blocked  H. EL-BEHEIRY  82  A Control  ACh  ACh  B  F i g . 24 D e p r e s s i o n by i s o f l u r a n e (IFL) of responses t o a c e t y l c h o l i n e (ACh; 150 nA f o r 18s i n A , 100 nA f o r 20s i n B) i n 2 neurons (A, V = - 7 0 mV; B, V = -68 mV). TTX (1.5 nM) was a p p l i e d i n A. Middle records (A,B) were o b t a i n e d a t 9 min o f IFL a p p l i c a t i o n . m  m  H. EL-BEHEIRY  83  15 mV  F i g . 25 D e p r e s s i o n by A l t h e s i n (AL) of responses t o ACh (125 nA f o r 9s i n A , 95 nA f o r 25s i n B) i n 2 neurons (A, Vm = -72 mV; B, V = -70 mV). N e g a t i v e v o l t a g e d e f l e c t i o n s ( t h i c k r e c o r d i n B) r e p r e s e n t t e s t s f o r changes in input resistance. TTX (1.5 nM) was a p p l i e d i n B. M i d d l e r e c o r d s were o b t a i n e d a t 8 min (A) and 6 min (B) o f AL a p p l i c a t i o n . m  H. EL-BEHEIRY  84  20 mV  F i g . 26 D e p r e s s i o n by i s o f l u r a n e ( I F L ) o f r e s p o n s e s t o g l u t a m a t e ( G l u ; 100 nA f o r 5s i n A, 95 nA f o r 8s i n B) i n 2 neurons ( V = -70 mV i n A and B ) . N e g a t i v e v o l t a g e d e f l e c t i o n s ( t h i c k r e c o r d i n B) r e p r e s e n t t e s t s f o r changes in input resistance. TTX (1 nM) was a p p l i e d i n B. M i d d l e r e c o r d s were o b t a i n e d a t 9 min (A) and 10 min (B) o f IFL a p p l i c a t i o n . m  B  Control  Glu  AL  NMDA  NMDA  IFL 2 MAC  100^M  Recovery  Fiq 27 A n a e s t h e t i c d e p r e s s i o n of responses t o G l u (35 nA) and NMDA (55 nA f o r 5s i n A , 110 nA f o r 15s i n B) i n 2 neurons ( V = -75 mV i n A, -69 mV i n B ) . R e s i s t a n c e t e s t p u l s e s were a p p l i e d o n l y i n B. TTX ( 1 . 5 pM) was a p p l i e d i n A . M i d d l e t r a c e s were o b t a i n e d a t 10 mm (A) and 9 min (B) o f a n a e s t h e t i c a p p l i c a t i o n . Voltage c a l i b r a t i o n : 20 mV i n A , 30 mV i n B. m  H. EL-BEHEIRY  86  F i g . 28 P o t e n t i a t i o n o f Glu a c t i o n s and a t t e n u a t i o n of ACh responses by i s o f l u r a n e (1MAC) i n an a n t e r i o r c i n g u l a t e neuron. Recovery was e v i d e n t a f t e r 16 m i n . V was k e p t c o n s t a n t a t -72 mV by c o n t i n u o u s D C - i n j e c t i o n . m  H. EL-BEHEIRY  Because to  o f t h e t h e poor  iontophoretically  sensitivities  applied  NMDA  of  observed  the  neurons  in  the  (layers  presence  87  IV  of  and  V)  external  +2 Mg  (2 mM),  a full  dose-response  these experiments.  Nonetheless,  depolarizations  a  in  relationship  both a n a e s t h e t i c s  dose-dependent  t h e same e x t e n t as t h e G l u - i n d u c e d Application  of  halothane  NMDA- and A C h - i n d u c e d isoflurane  manner  be c o n s t r u c t e d  in  a t t e n u a t e d t h e NMDA-evoked  ( F i g . 27)  and  to  approximately  responses.  (1.5 MAC;  responses  not  in  6  a manner  neurons)  suppressed  similar  to  that  the  Glu-,  observed  with  administration.  5.1.2.2 (5 neurons;  Responses  F i g . 29A)  by GABA a p p l i c a t i o n s . GABA by ~15%. depolarizing  t o GABA.  Isoflurane  administration  1.5 MAC  responses  However,  t h e responses  2 MAC i s o f l u r a n e  suppressed  evoked to  A d m i n i s t r a t i o n of i s o f l u r a n e o r A l t h e s i n s l i g h t l y reduced t h e to  GABA  in  the  25-100 LIM A l t h e s i n , F i g s .  low  dose  29B and 3 0 ) .  range  i s o f l u r a n e and 200 nM A l t h e s i n were a p p l i e d ( F i g . Selectivity  in  the  depression  ( 0 . 5 - 1 . 5 MAC  The maximum  r e s p o n s e s was not more t h a t 40% even when doses as  5.1.3  at  had no e f f e c t on t h e h y p e r p o l a r i z i n g  responses  isoflurane; i n these  could  depression  h i g h as 2.5 MAC  31).  of  responses  to  transmitter  substances 5.1.3.1 neurons  Anaesthetic  with  transmitters.  a n a e s t h e t i c a p p l i c a t i o n produced r e d u c t i o n s  ACh and G l u ( T a b l e 1 ) . to depression apparent  interactions  in  approximately  However,  by e i t h e r neurons equal  much more pronounced  where in  or A l t h e s i n .  initially,  amplitude  in the responses to  a c e t y l c h o l i n e a c t i o n s were more  isoflurane  (Figs.  with isoflurane  these 29B  This  than a f t e r  32).  The  Althesin  most both  susceptible  s e l e c t i v i t y was  transmitter  and  In  responses  most were  selectivity  application  32 ) and was not o b s e r v e d on p e r f u s i o n of t h e v e h i c l e , cremophor EL.  was (Fig.  In 7  H. EL-BEHEIRY  88  F i g . 29 E f f e c t s o f i s o f l u r a n e ( I F L ) a p p l i c a t i o n on e q u i - a m p l i t u d e responses t o GABA (100 nA f o r 5s i n A, 75 nA f o r 6s i n B ) , G l u , and ACh i n 2 neurons ( V = - 7 5 mV i n A, -72 mV i n B ) . TTX (1 M) was a p p l i e d i n A. Resistance t e s t p u l s e s were a p p l i e d o n l y i n B. G l u (85 nA) and ACh (90 nA) were a p p l i e d i n B. M i d d l e t r a c e s i n A and B were o b t a i n e d a t 9 min o f IFL application. V o l t a g e c a l i b r a t i o n : 10 mV i n A, 20 mV i n B. m  M  30 mV 20s m i  F i g . 30 E f f e c t s o f A l t h e s i n a p p l i c a t i o n on the ACh, GABA and Glu-evoked responses (ACh 70 nA f o r 23s; Glu 90 nA f o r 10s; GABA 110 nA f o r 1 0 s ) . Note t h a t , i n i t i a l l y , a prominent hyperp o l a r i z i n g response t o ACh was p r e s e n t . A n a e s t h e t i c a p p l i c a t i o n depressed the d e p o l a r i z a t i o n induced by ACh, s l i g h t l y a t t e n u a t e d t h e Glu-response and had no e f f e c t on t h e response t o GABA.  H. EL-BEHEIRY  Glu  ACh  90  GABA  Isoflurane (2 MAC I ZnTlfrTTm^^  wrrrTrrrmrHTT  t17  25s 20 mV  F i g . 31 D e p r e s s i o n o f ACh, G l u and GABA responses by h i g h dose o f i s o f l u r a n e i n neuron (Vm = -68 mV ; ACh 85 nA f o r 10s; G l u 100 nA f o r 5 s ; GABA 75 nA for 8s).  H. EL-BEHEIRY T a b l e 1.  A n a e s t h e t i c d e p r e s s i o n of neuronal transmitter  Anaesthetic applied  Glu (n = 65)  Isoflurane  18/18  30/40  8/10  Althesin  15/16  25/32  7/9  Halothane  3/4  5/7  Depression  was  anaesthetic. neurons  The t o t a l  were t e s t e d t o  The a n a e s t h e t i c and 0.5-2  defined  GABA evoked  NMDA (n = 15)  Hyperpolari zation (n = 9 )  Depolari zation (n = 29)  4/9  10/17 11/15 2/4  as  >15% a t t e n u a t i o n  number  of  Glu and t o  the at  halothane.  of  neurons least  doses were 0 . 5 - 2 . 5 MAC f o r  MAC f o r  responses to  substances  D e p o l a r i z a t i o n evoked by ACh (n = 36)  91  one  control  investigated other  isoflurane,  responses was  65,  transmitter 25-200 nM f o r  by  the  i.e,  all  substance. Althesin  H. EL-BEHEIRY  A , TOO nA i n B ) . Resistance test pulses calibration: 20 mV i n A, 10 mV i n B.  were a p p l i e d o n l y  i n A.  92  Voltage  H. EL-BEHEIRY neurons,  low  depression  dose  administration  of  isoflurane  or  Althesin  of the 61 u - d e p o l a r i z a t i o n and sometimes p r o l o n g e d  t h e G l u response  (cf. Fig.  93  produced  no  the d u r a t i o n  of  32B).  When GABA, Ach and/or G l u were a p p l i e d a l t e r n a t i v e l y t o the same neuron, the  additional  alter  a p p l i c a t i o n of  the d e p o l a r i z i n g  isoflurane  e f f e c t s of  or A l t h e s i n  GABA,  slightly  markedly a t t e n u a t e d t h e r e s p o n s e s t o Ach ( F i g s . 5.1.3.2 produced  by  response  usually  after  Time  course  isoflurane  "8-12  was  min of  Althesin  suppressed  anaesthetic  after  ~3  detectable  a t t e n u a t i o n o f t h e Glu r e s p o n s e s .  time  suppressed  courses  responses.  that  Full  the  were  Ach  were  responses similar  recovery  u s u a l l y was observed  to  several  relationships responses 35. 4  applications  apparent were  by  the  transmitter  maximally  33). at  In  5  neurons,  30-90s,  Administration of  the  anaesthetic  reduced  i.e., of  19  the  before  isoflurane,  neurons)  reductions  with  in  Glu  depolarization  33).  relationships.  of  either  of  Glu  The  limited.  The  Such  0.9  are  shown  an averaged  anaesthetic.  responses.  i n a comparison  substances  represents  of t h e Ach r e s p o n s e s was  more  (Fig.  was  Ach-evoked  dose-response  f o r the a c t i o n s o f i s o f l u r a n e and A l t h e s i n on t h e j u s t - m a x i m a l  Each p o i n t on the c u r v e s  suppression  and  minutes e a r l i e r than a complete r e c o v e r y o f t h e  Dose-response  evoked  depression  Glu  depressions  The  from a n a e s t h e t i c e f f e c t s on the G l u  Ach responses t o c o n t r o l a m p l i t u d e ( F i g . 5.1.3.3  and  evident  the  of  The  onsets.  t o NMDA (15  to  those  actions.  min,  application  significantly  29B-31).  rapid  in  or  responses  had  not  reduced  anaesthetic  reductions  Althesin  the  or  of  did  The  EC^  in  0  for  was  less  the 1.9  and by  isoflurane MAC  although  pronounced  34  produced  selectivity  of Glu w i t h t h e NMDA r e s p o n s e s , selectivity  Figs.  depression  MAC compared w i t h  differences  in  in  for  also the the  the were data case  H. EL-BEHEIRY  94  100  75-• - • GABA (n=5) A - A Glu (n=6) o-o ACh (n=6)  50--  25--  Isoflurane (1  MAC)  12  18  TIME (min)  F i g . 33 Time c o u r s e s f o r a n a e s t h e t i c d e p r e s s i o n o f j u s t - m a x i m a l responses t o GABA, G l u and ACh i n the p r e s e n c e of TTX (1 LIM) . Two o r 3 t r a n s m i t t e r substances were a p p l i e d a l t e r n a t i v e l y t o each neuron. E r r o r bars i n d i c a t e * S.E. mean.  H. EL-BEHEIRY  100  T  O Ach  95  V NMDA  80--  c o o N  60--  40--  _o o  OL CD Q  20--  ~XD  CU  o >  100-r  c o  CO  cu  CL CD Q  1-0  2.0  0.5  1.0  2.0  Isoflurane Concentration (MAC)  F i g . 34 P o o l e d d a t a show dose-response r e l a t i o n s h i p s f o r i s o f l u r a n e - i n d u c e d d e p r e s s i o n s o f d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r s u b s t a n c e s . Each p o i n t on t h e c u r v e i s t h e mean response t o a t l e a s t 4 a p p l i c a t i o n s (n = 2 2 ) . Symbols (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t p < 0.05. O v e r a l l s i g n i f i c a n c e was determined by ANOVA. E r r o r bars i n d i c a t e *S.E. mean.  H. EL-BEHEIRY  96  F i g . 35 Dose-response r e l a t i o n s h i p s for Althesin-induced depression of d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r s u b s t a n c e s . Each p o i n t on the curve i s t h e mean response t o a t l e a s t 4 a p p l i c a t i o n s (n = 2 0 ) . Symbols (*) indicate significant difference from c o n t r o l at p < 0.05. Overall s i g n i f i c a n c e was d e t e r m i n e d by ANOVA. E r r o r bars i n d i c a t e * S . E . mean.  H. EL-BEHEIRY  of  Althesin  responses maximal  where  and  the  90  EC s  uM f o r  reductions  in  were  5 0  the  the  75  nM  depression  depolarizing  for  of  the  Glu  depression  responses  responses  to  of  (Fig.  97  the  ACh  35).  The  GABA a p p l i c a t i o n  were  o n l y about 40% at 2.5 MAC i s o f l u r a n e o r 200 uM A l t h e s i n . The e f f e c t s ~ED  5Q  were  of a t r a n s m i t t e r substance  36A). more  of a n a e s t h e t i c s  the by  depolarizations anaesthetics.  than  were This  by  more  response  Althesin  susceptibility  to  also  a n a e s t h e t i c e f f e c t s on the j u s t - m a x i m a l  5.1.3.4 by  ACh.  for  ("EDJJQ)  s,  and  6s  then  Althesin  were  3 of  additive  appeared  to  depolarization. administered, anaesthetic  was  can  the  there  be  disproportionate  a  the be  also  was  (ACh/Glu)  depressant  the  by  the (Fig.  depressed ACh-evoked  actions  of  both  investigations  to  no  (Fig.  of  potentiation  in  7  of  of  ACh  the  from Glu  of  neurons  anaesthetic seen  36B).  (~ED  5  Q  applying  lasting  )  (isoflurane Figs.  i.e.,  change  Glu-responses  by  in  37 the  magnitude  of  evident.  reduced the G l u r e s p o n s e s  4,  and  38,  discharge the  o r 75 nM A l t h e s i n was longer  120-180  in  and  Glu  the  depolarizations  the  which  which  combined  additionally  Note  about t h e same time as t h e ACh e f f e c t s from t h e a n a e s t h e t i c 5.2  was  although  revealed  the  qualitative  MAC i s o f l u r a n e  enhancement  applications  on  effects  but  this  was  i n c a s e s where  r e s p o n s e s t o ACh, G l u and GABA w h i c h  applications  As  enhanced  When 1.5  was  examined  applying  neurons). ACh  effects  longer  concomitantly  in  application  possibility during  the  i n t h e same neurons  Anaesthetic  This  ratio  application,  susceptible  were a p p l i e d f o r comparison  quantified  had been a p p l i e d t o t h e same neuron  acetylcholine/glutamate  isoflurane  further  that  such  recovered  at  depression.  Discussion 5.2.1  Responses t o t r a n s m i t t e r and r e l a t e d a g e n t s .  no fundamental d i f f e r e n c e s between t h e r e s p o n s i v e n e s s  A l t h o u g h t h e r e were  of n e o c o r t i c a l  neurons  H. EL-BEHEIRY  Isoflurane (1.5 MAC)  98  Althesin (75 /xM)  Fig. 36 Selectivity in i s o f l u r a n e and Althesin-evoked depression of neuronal responsiveness t o ACh, G l u and GABA. (A) ACh/Glu ratio of d e p o l a r i z i n g responses was d e p r e s s e d by a n a e s t h e t i c a p p l i c a t i o n s i n d i c a t i n g g r e a t e r s u s c e p t i b i l i t y of the ACh r e s p o n s e s . E r r o r bars show ± S . E . mean. Symbols (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t p < 0 . 0 5 . (B) p o o l e d d a t a from 12 neurons i n which a t l e a s t 2 t r a n s m i t t e r substances evoked j u s t - m a x i m a l response. Error bars show ± S . E . mean. Post-hoc comparison t e s t was u s e d . Symbols (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t p < 0.05 as d e t e r m i n e d by S t u d e n t ' s t - t e s t . Overall significance was determined by ANOVA.  H. EL-BEHEIRY  99  IFL 11.5 MACI  ACh  25 mV  ACh  F i g . 37 Continuous r e c o r d of i s o f l u r a n e ( I F L ) - i n d u c e d d e p r e s s i o n of the ACh-potentiation of Glu-actions. Depolarizations evoked by Glu (cf. n e g a t i v e a r t e f a c t s a t o n s e t of 70 nA c u r r e n t ) and ACh (50 nA) are ~ 50% of t h e i r maximal r e s p o n s e s i n t h i s neuron. IFL was a p p l i e d f o r 5 min ( t h i c k bar).  H. EL-BEHEIRY  Glu  100  ACh  Althesin 1 0 0 M M  ACh  30mV  ACh  60s  Fig. 38 Althesin-induced depression of the ACh-potentiation of Glu-actions. D e p o l a r i z a t i o n s evoked by G l u (60 nA) and ACh (40 nA) were d e p r e s s e d by A l t h e s i n a p p l i c a t i o n a t 8 m i n ; r e c o v e r y was e v i d e n t a f t e r 15 min ( l o w e r t r a c e ) .  H. EL-BEHEIRY  in these  investigations  in  areas  other  of  and t h e  the  CNS  sensitivities  (Krnjevic  1974b)  of  to  other  the  neuronal  5.2.1.1 resistance in  Acetylcholine.  The  (Krnjevic  et a l .  1971b)  In n e o c o r t i c a l s l i c e p r e p a r a t i o n s , t h a t ACh evoked a r a t h e r firing  depolarization  induced by ACh were d e s c r i b e d p r e v i o u s l y  decerebrate  that  was  conductance.  small  preceded  The i n i t i a l  and P r i n c e  long-lasting  by  a  for  brief  could  present  be  blocked  investigations,  application  from  two  both  types  activating  an i n t e r n e u r o n  assemblies  having  intertip  types of  is  by  likely  to  distances  responses  placements.  be l e s s  of  whereas  independent  iontophoretic  pipette,  w h i c h c o u l d be b l o c k e d w i t h TTX response.  The " a f t e r b u r s t "  of  evoked  and was  synaptic  a  and  an  observed The  ~50 um because  observed,  receptors  e t a l . 1983)  of  o r spontaneous  Not a l l t h e neurons applications Glu.  nerve  (~70%  were  terminals  1974).  the  increased  ACh  always activity  of  because In  the  after  ACh of  electrode  transmitter  With such  is  assemblies  application  short-lasting  (Ropert  burst  possibility  from  an  hyperpolarization  f o l l o w e d by  a  depolarizing  recorded only  occasionally  i n the p r e s e n c e of TTX ( c f . F i g . 17C) c o u l d be due t o p r e s y n a p t i c nicotinic  observed  w i t h t h e compound  only  were  1978).  application.  were  (Puil  depolarizations  (Woody e t a l .  micropipette  TTX  e j e c t e d m o s t l y near t h e soma of t h e neuron slow  neurons  a t t r i b u t e d to a c t i v a t i o n  concomitant  electrode  input  neocortical  hyperpolarization  of  increased  depolarization with spike  h y p e r p o l a r i z a t i o n was  selectively  substances,  (1986) a l s o  an i n t e r n e u r o n by t h e ACh " p u f f e d " from an independent it  types  investigations.  and  and awake c a t s  McCormick  cell  transmitter  some q u a l i t a t i v e d i f f e r e n c e s have been i d e n t i f i e d by t h e s e  101  and  Krnjevic  actions  1982;  on  Rovira  release of e x c i t a t o r y t r a n s m i t t e r s .  (presumably responsive)  P a r t of t h i s A C h - i n s e n s i t i v i t y  pyramidal despite  c e l l s ) , were r e s p o n s i v e their  high  t o ACh  sensitivities  to  c o u l d be e x p l a i n e d by t h e damage of t h e  H. EL-BEHEIRY  neurons  produced  particularly  by  in  view  resistance  by  differences  between  intact  or  provide The  of  the  decreasing  be  secondary  messenger  D u t a r and N i c o l l  in  period (e.g., acute  ACh  the  to  recording  increase  conductance in vitro  where  the  for  and  pathways  effects  of  to  a  ACh  applied  long-term  electrode,  neuronal K .  recorded  are  in  the  undisturbed  may  i n 30% of t h e on  input  Functional  +  those  the  neurons.  neurons  modulation  systems ( c f . Woody and Gruen 1988;  of  (cf.  muscarinic  Muller et a l .  1988;  1988). Glutamate  the  qualitatively  by  the i n s e n s i t i v i t y observed  attributable  5.2.1.2 feature  of  recorded  long-lasting  may  action  preparations  other reasons f o r  Results)  penetration  membrane  neurons  decerebrate  relatively  both  electrode  102  Glu-induced  and 3-4  and  and  responses  quantitatively hours).  N-methyl-D-aspartate.  despite  was the  An  their length  outstanding  consistency of  the  experimental  U n l i k e G l u , NMDA-evoked d e p o l a r i z a t i o n s  long-term  forms  of  desensitization,  probably  both  exhibited  due  to  the  +2 presence 1987).  of The  external occasional  and ACh ( e s p e c i a l l y assemblies)  Mg  could  the g l u t a m a t e r g i c  (Mayer  difficulty  i n the c a s e be  explained  in  Westbrook  inducing  by  the  known  1987;  equivalent  of a p p l i c a t i o n s  and c h o l i n e r g i c  example, t h e l o c a t i o n s o f h i g h  and  MacDonald  et a l .  responses  to  from t h e compound  topographical  ( i n t h i s case, muscarinic)  s e n s i t i v i t y or the " h o t  Glu  electrode  distribution receptors.  spots"  of For  f o r G l u may be  on d e n d r i t e s whereas t h o s e f o r ACh a r e l o c a t e d m a i n l y on the soma. , produced  5.2.1.3  Y-aminobuty r a t e .  hyperpol a r i z i n g  responses electrodes  could and  be were  and  evoked readily  GABA a p p l i c a t i o n s  depolarizing only  by  blocked  GABA by  to neocortical  responses.  The  application  bicuculline  neurons  hyperpol a r i z i n g  from  (50 uM);  the  compound  these  could  H. EL-BEHEIRY  result  from  interactions  with  somatic  GABA  receptors  A  (cf.  103  Bormann  1988).  The d e p o l a r i z i n g r e s p o n s e s observed e x c l u s i v e l y w i t h t h e s e p a r a t e a p p l i c a t i o n and r e c o r d i n g e l e c t r o d e s are p r o b a b l y a t t r i b u t a b l e t o s t i m u l a t i o n o f subpopulation of receptors pyramidal  neurons  (cf.  f o r GABA t h a t are l o c a t e d on t h e d e n d r i t e s of t h e  Alger  and  Nicol  GABA have been r e p o r t e d i n t h e s p i n a l and  Gynther  (Andersen and  1987)  and  e t a l . 1980;  Perreault  after  1982).  Blaxter  dendritic  and  Depolarizing  cord (Barker  Carlen  responses  and Ransom 1978b;  application  Thalmann e t a l . 1981;  1987;  another  in  the  In  cortical  Curtis  hippocampus  A l g e r and N i c o l 1  1988).  1982;  Avoli  neurons  t y p e o f response  can be b l o c k e d by b i c u c u l l i n e and i s m e d i a t e d m o s t l y  increase  conductance  1979,  in CI"  1982;  1986b).  Andersen  In  et a l .  the p r e s e n t  of  these  shifted  (>15 m i n ;  responses by  presence of TTX  (cf.  mechanisms  the  response  and evoked  by  Thalmann  be  1981;  F i g . 21C). of was  in  the  Despite  Moreover, when  range  a p p l i c a t i o n o f G l u t o t h e same neuron.  resting -100  to  of In  mediating  were  "shunting" several  neurons  spike  of  GABA may, u n d e r l i e  phenomena d e s c r i b e d e a r l i e r by P u i l slab investigations release  of  H  Schwartz  1967).  +  a  paradoxical  with  ionic the by  response  excitatory  e t a l . (1974) i n t h e i s o l a t e d n e o c o r t i c a l  or r e p o r t e d f o l l o w i n g s t i m u l a t i o n o f c o r t i c a l  concomitantly  the  activities  to  actions  in  elicited  and f a l l i n g phases o f t h e d e p o l a r i z i n g  similar  was  response,  were observed on t h e r i s i n g GABA;  only  potential  to the  spikes  al.  prolonged  -30 mV  this  et  potential  the u n c e r t a i n i t i e s as  GABA-receptor capable  of  by an  Misgeld  a reversal  the  this  and N i c o l 1  the GABA d e p o l a r i z a t i o n s  attained  injection  GABA  Alger  LIM) of b i c u c u l l i n e and t h e n o n l y a f t e r  not  subtype  1967;  et a l .  2ml/min f l o w r a t e ) .  could  DC-current  1980;  and Schwartz  investigations,  b l o c k e d by l a r g e doses (>100 applications  (Krnjevic  to  the  GABA  ejections  (cf.  neurons  Krnjevic  by and  H. EL-BEHEIRY  5.2.2  Anaesthetic-induced  extracellularly  applied  transmitter  (1963a) found many y e a r s sensitivity cerveau  ago,  in  agents.  that cerebral  preparations,  the  neocortical  only  a  anaesthetic-induced neurons  to  the  cortical  few  Krnjevic  transmitters  Phi 11is  i n comparison  investigations in  and  of  neurons showed a lower  animals  alterations  various  chemosensitivity  Although  t o ACh i n b a r b i t u r a t e - a n a e s t h e t i z e d  i soli  explored  alterations  104  to  reportedly  the  the have  responsiveness  and  related  substances.  Subsequent r e p o r t s by K r n j e v i c and h i s group r e p o r t e d t h a t a wide v a r i e t y intravenous  and  volatile  actions  ACh  while  of  decerebrate Catchlove  animals  et a l .  anaesthetics  the  G l u - and  (Krnjevic  1972;  selectively the  and  However,  this  preferential  attenuation  has  been  confirmed  by  other  Bazil  and  not  Crawford  1970;  thiopental  cf.  elicited  by  (5-hydroxytryptamine, of d e c e r e b r a t e c a t s extracellular  abovementioned effects  1975;  of ACh  investigators Minnemann  reports  Another  i n t a c t ascending as  anaesthetics  noradrenaline  well  is  the  neurons e x c i t e d by  may  inhibitory to  be  uniformly, ACh  the  or  unit  the  to  with  varying  and e x c i t a t o r y pathways  the  variability  in  the  on t h e c e r e b r a l blood f l o w of t h e i n t a c t  cells 1966;  monoamines neurons  i n the in  interactions  applications. of the  degrees  r a d i a t e to the effects animals.  or  excitatory  activity  the  transmitter  the  1978).  in neocortical  discharge  in  1966;  halothane  A major d i s a d v a n t a g e  problem  related  et a l .  and C u r t i s  Moreover,  of  preserved  neocortical  f o r the d i s s i m i l a r i t i e s with the r e s u l t s  reason  as  on  of  excitatory  and K r n j e v i c  (Crawford  1989).  for  inherent  were  Puil  and i s o p r e n a l i n e )  techniques  the  Krnjevic  actions  applications  recording  may p r o v i d e a reason  cortex  Puil  (Johnson e t a l . 1969).  from nearby  studies.  1963b, c ;  a p p l i c a t i o n have been shown t o d e p r e s s ,  responses  of  GABA-responses  and Phi 11 i s  Krnjevi6  depressed  of  of  use the of This  present  that  the  cerebral different  Unfortunately, effects  of  levels  of  Despite  serotonin  inhalational  Smaje 1976).  Yamamoto  responses  of  suppressed  1985).  Glu  effects  olfactory  of  bulb  to  are  less  affected  application  of  ACh-facilitation S t e i n e r 1963) by  and Dray than  (Brooks  degrees  by  unit  Phillis  noradrenaline  in vivo  and  experiments,  and Smaje  to  ACh  1986).  applied  P-evoked  or  activity  of  administered  in  Barbiturates  and  i n t h e thalamus  not doses  various (Phillis  Spencer and H a v l i c e k  neuronal  excitations  cat  and  e x h i b i t a s e l e c t i v e decrease  agents  medullary  in vivo  are  Higher  intermittently with  volatile  neurons  and d i d not have any e f f e c t  Interpeduncular  substance  1976;  (Sawada  depolarizations  neurons  monoamines  responses  during  (Sastry  and Renshaw c e l l s ( B i s c o e and K r n j e v i c 1963)  anaesthetics  and  barbiturates  GABA-elicited  et a l .  1973).  intravenous of  depress  spontaneously  (McCance  in  at  cerebellar  ka and NMDA i n hippocampal CA1  varying  G l u - and  chemosensitivities  and Glu ( B r a d l e y  on  i n v i t r o (Richards  B r a i n s t e m neurons and s p i n a l motoneurons  their  the  i s o f l u r a n e a p p l i c a t i o n i n low doses.  GABA  neurons  selectively  and T e b e c i s 1967) and b a s a l g a n g l i a (Bloom e t a l . 1965;  in  In  ACh,  e t h e r s a t t e n u a t e ACh- and not G l u - i n d u c e d d i s c h a r g e s  1974).  other  actions  depressed o n l y t h e G l u - r e s p o n s e s to  of  about  G l u - f i r i n g and p o t e n t i a t e A C h - e x c i t a t i o n r e c o r d e d  However,  responses  the  barbiturates  the  the  available  mammals.  extracellularly  i n the o l f a c t o r y c o r t e x  o f t h e s e agents the  of  Responses t o G l u , Q u i s ,  are  of  attenuating  suppression  a f f e c t e d by h a l o t h a n e o r  on  neuraxis  recorded  agents depress  extracellularly  vitro  without  is  105  responsiveness  preparations,  units  the  on u n i t  central  cat  neuronal  information  on the c h e m i c a l  the  excitation  discharging  in  in  i n vivo  ACh-evoked  1964).  incomplete  anaesthetics  different cortex  only  H. EL-BEHEIRY  t o ACh  anaesthetic 1978).  (Salmoiraghi is  preparations.  The and  not a t t e n u a t e d Although  the  H. EL-BEHEIRY  available  evidence  possibility suspected the  may  be  interpreted  that anaesthetics  transmitters  production  of  on  the  only  with  some  106  uncertainties,  the  have s e l e c t i v e e f f e c t s on the s e n s i t i v i t i e s  subcortical  anaesthetic  neurons  state  that  should  have  be  some  relevance  subjected  to  to to  extensive  re-investigation. In  the  present  and TTX-blockade modifications  of  in  intact  the  use  genesis  chemical  of  the  f o r the  in vitro  slice  investigations  responsiveness  of  the  of  neocortical  An  these  results  interesting  with the  compared  changes  of  to  the  synaptic  observation  G l u - or  blockade  in  the  present  studies  of  the  transmitter  agents  channel-receptor  NMDA-evoked  depolarizations.  TTX  and  absence  complex  be or  due  on  the  i n the passive  chemosensitivity could  in  of  to  the  the  anaesthetic of  Gruener  Flanigan  Lechleiter  and B r e t t 1987;  depression maximum  1984;  produced  number  stabilization  of of  by  et a l .  Ikemoto  closed  1986;  may  activatable state  of  on  Arimura  by the  and  ACh.  from This  channel  or  a  the  Under  the  excitatory the  ionic  messenger  transduction. (Lechleiter  Ikemoto  suggest t h a t t h e  result  is  significant  secondary  on d i f f e r e n t systems  e t a l . 1988)  anaesthetics  channels the  studies  the  effects the  from  properties,  to  system r e s p o n s i b l e f o r the a g o n i s t / r e c e p t o r b i n d i n g impulse Recent p a t c h - and v o l t a g e - c l a m p  of  membrane  neurons  modulation  should  ACh-induced  the  by  induced by t h e a n a e s t h e t i c  suppression  and  neurons  information obtained  g r e a t e r a t t e n u a t i o n by i s o f l u r a n e or A l t h e s i n a p p l i c a t i o n , of t h e  conditions  anaesthetic  i n neuronal m e t a b o l i s m i n v i t r o and i n v i v o a l s o  i n comparing  animals.  responses  preparation  i n h i b i t o r y or e x c i t a t o r y influences external to the recorded  Differences  be c o n s i d e r e d  Na-spike  the  reduced p o s s i b l e neuron.  experiments,  1986;  cholinergic  decrease  in  the  may  be  due  to  a  to  an  allosteric  H. EL-BEHEIRY  modification constants  of  the channel  governing  proteins  transitions  leading  between  to  the  a  open  n o r m a l l y o c c u l t c l o s e d s t a t e s , w i t h a subsequent ionophore.  Binding  studies  have  bind with the ACh-receptor  binding  or  of t h e s e i n vivo found  s t a b i l i z e the  actions situation  to  states  of  protein  lower  corresponds  G protein-coupled agonist  complexes  for  affinity  guanine  the  rate  state  one  or  more  excessive  s t a t e of to the  (Young and Sigman 1981).  convert  of  a l l o s t e r i c a l l y and  high-affinity  probably  shortening  shown t h a t a wide range  thetics  to  of  general  i n h i b i t the  addition,  lower  nucleotides  f l i c k e r i n g of t h e  the r e c e p t o r ;  and - u n c o u p l e d  and  and  halothane  affinity  (Aronstam  specific  et a l .  outcome  state  muscarinic  the  anaes-  the  non-desensitized  In  107  in  has  the been  receptor of  to  receptor-G  1986;  Dennison  e t a l . 1987; Anthony e t a l . 1989). On the o t h e r hand, t h e d e p r e s s i o n  of the G l u - i n d u c e d  depolarizations  i s o f l u r a n e and A l t h e s i n c o u l d be a t t r i b u t e d i n p a r t t o an i n c r e a s e d of Glu to  its  activatable  receptors  and p a r t l y t o a decrease  receptor-channel  complexes  which  of e i t h e r a blocked-closed  or d e s e n s i t i z e d  However,  supportive  i n t h e absence  suggestions (1989) [Ca of  of  remain h i g h l y  observed  that  ].. i n c u l t u r e d hippocampal isoflurane  and  halothane  state  More  attenuated  neurons.  most  would  t h e maximal  result  (cf.  biochemical  speculative.  isoflurane  in  et a l .  pentobarbital and  high  of  t h e GABA-evoked  involve  a blockade  of  enflurane  in  of  Ca-channels of  receptors  Ca-channels. depolarizations  i n doses h i g h e r than 300 nM, a n a e s t h e t i c  doses  Baimbridge  increase  a c t i v a t e d by i n t e r a c t i o n s of Glu w i t h e i t h e r Quis o r NMDA t y p e s  A suppression  such  They c o n c l u d e d t h a t t h e a c t i o n s  likely  and a d i r e c t b l o c k a d e o f v o l t a g e - g a t e d  1988).  data  and  Glu-evoked  of  formation  and p a t c h - c l a m p Puil  affinity number  the  Ikemoto  recently, the  in  by  (Connors  1980;  has  been  doses o f  Simmonds  1981;  reported  for  alphaxalone, Cullen  and  H. EL-BEHEIRY M a r t i n 1982; effects  P a r k e r e t a l . 1986;  observed  Possibilities trations  of  previously  Brooks e t a l . 1986).  and  in  include the plugging the  anaesthetic,  endplate channels  (Adams 1976)  the  present  to  that  neurons  in vivo  and P h i l l i s  The observed d o s e - r e s p o n s e  relationships  of e x c i t a t o r y and  the  animal  a  suppression responses.  and This  ending is  continuum  with  o f CNS  more s e l e c t i v e a n a e s t h e t i c The r e s u l t s o f t h e s e tions  unit discharge  to  inherent  the  at  concen-  ACh-activated on t h e  results  on mammalian  neocortical  Catchlove  al.,  et  1972).  inhibitory transmitter interference  attenuation  actions  beginning  of  the  with  Glu  the  (and  AChNMDA-)  t o the g e n e r a l i z e d c o n c e p t o f d e p r e s s i o n  neurons  during  anaesthesia,  and  in  suggests  of  much  actions. experiments  in o l f a c t o r y c o r t i c a l  depress  of  complete  in contrast  the c h e m o s e n s i t i v i t i e s  by h i g h  clear.  a l l u d e t o the p o s s i b i l i t y t h a t  depressions are  ago  1963a,b,c;  anaesthetic intact  observed  not  are c o n c o r d a n t w i t h t h e  performed many y e a r s  (Krnjevic  are  molecules.  Q u a l i t a t i v e l y , the present investigations e x t r a c e l l u l a r studies  studies  o r t h e o c c u p a t i o n of t h e a c t i v e s i t e s  GABA r e c e p t o r by the a n a e s t h e t i c  of  The r e a s o n s f o r t h e  of t h e c h l o r i d e c h a n n e l s  similar  108  neurons  are not i n agreement w i t h the where  inhalational  evoked by ACh a p p l i c a t i o n (Smaje  differences  in  the  membrane  anaesthetics 1976).  In  e x c i t a b i l i t i e s of  observadid  not  addition  neocortical  and o l f a c t o r y neurons,  t h e low doses of a n a e s t h e t i c s a p p l i e d t o t h e o l f a c t o r y  cortical  explain  slices  investigations, administered  in  may  halothane, doses  of  diethyl <0.5  c o n c e n t r a t i o n s o f t h e s e agents f i r i n g r a t e o f t h e neurons  the  MAC  discrepant ether values  results.  and  In  the  trichloroethylene  for  rodents.  olfactory had  been  Increasing  the  i n the atmosphere o f t h e s l i c e suppressed  ( c f . F i g . 4 i n Smaje  1976).  the  H. EL-BEHEIRY  In  conclusion,  in vitro in  a  the  preparation  selective  chemosensitivities  are  and  reduced  gradual  by  of  neocortical  applications  manner;  these  of  begin  w i t h c o m p l e t e a t t e n u a t i o n of t h e A C h - and G l u - r e s p o n s e s anaesthetic the  and  ED^Q  ACh-induced  responses  Glu-depolarizations, molecules such an and/or  to  EDg^s  their  may  respectively. at  low  reflect  sites  This  doses,  different  (membrane  the  secondary  messenger  r e c e p t o r b i n d i n g impulse  6  6.1  systems  the  Althesin and  end  approximately at  the  attenuation  for  action.  the  responsible  of  of  the  anaesthetic  The mechanism  i n t e r f e r e n c e may be r e l a t e d t o t h e c h a n n e l - r e c e p t o r to  or  preservation  affinities of  in  w i t h ACh  selective  with  receptors)  neurons  isoflurane  effects  109  complex  for  the  of  protein agonist-  transduction.  EFFECTS OF HYPOMAGNESIA ON TRANSMITTER AND ANAESTHETIC ACTIONS  Results 2+ The  effects  sensorimotor  of  a  neurons  reduction on  their  in  (±5.97; current durations neurons  These  neurons  ±S.D.) mV  and  injections of  less  revealed  (± 8 . 7 ) neurons  and  20.5  were  44.96  evoked  spikes  than  ms.  2  inward  (50-300 nA f o r 4-20  had  s)  to  were  extracellular  a  mean  V  and  m  ( ± 1 6 . 4 6 ) Mn, with The  R^  in  28  applications  of  amplitudes  (~15%)  resistance  of  current-voltage Applications  mV r e s p e c t i v e l y .  isoflurane  respectively.  produced mean d e p o l a r i z a t i o n s  increased  investigated  and a f t e r an a p p l i c a t i o n of  rectification.  (± 6.8)  either  ]  [Mg  responses  ACh, G l u and GABA b e f o r e , d u r i n g , Althesin.  the  The or  input  than  curves  in  of  ACh, 13.12  80 mV  and  85% o f  the  Glu  and  GABA  (± 3 . 6 ) ,  22.5  resistances  unaffected  -76.3  Intracellular  more  of  of  or  near  of  these  the  peak  H. EL-BEHEIRY depolarizing  response  induced  by  ACh.  Input  resistances  110  were  always  d e c r e a s e d (35-90%) by G l u o r GABA a p p l i c a t i o n s d e s p i t e the p r e s e n c e (n =  18)  or absence ( n = 10) of TTX ( 1 - 1 . 5 uM). 6.1.1 2+ [Mg  ]  Effects in  the  impurities  in  bath  nominally the  1987).  Perfusion  presence  or  neurons.  of  Mg-free  salts of  absence However,  application  used  the  the  (Mayer  of  the  b l o c k e r the  +  were  greatly  medium  and  spontaneous  enhanced.  TTX  After  perfusion  depolarizations present  of  ACh  (5/6  ( F i g . 39B).  On the  neurons)  a f f e c t e d during perfusion d e p o l a r i z a t i o n s evoked by  other  of  comparable t o the G l u responses solution  medium,  a brief  6/10 neurons 6.1.2 different  (10/10  a p p l i c a t i o n of  (Fig. 40). Effects  of  et a l . in  (3-5 mV)  the in  8 or  In the absence o f  the  synaptic  in  activities  nominally  spontaneous  slow  Mg-free  depolarizing  39A). and  GABA  i n Mg-free hand,  (10/10  neurons)  solutions  with or  responses  were  Glu  potentiated.  During  a  without  TTX  inconsistently The 40)  Responses t o NMDA t h a t were  continuous  NMDA evoked  induced  zero mM Mg. ( F i g s . 39B and  c o u l d not be o b t a i n e d d u r i n g  neurons).  trace  unchanged  background  incubation  of  solution,  either  with solutions containing Glu were e i t h e r depressed  unchanged o r , on o t h e r o c c a s i o n s ,  control  were  calculated  Sutor  hyperpolarization  the  periods  t h a t were reduced  1985;  respectively).  and  The  because  "Mg-free"  neurons  waves were r e c o r d e d i n 4 neurons ( c f . F i g . Applications  ~ l - 3 uM  this  ~ l - 3 hrs  abrupt  ACSF.  Westbrook  slight  d e c r e a s e d by ~10% (5/8 and 3/8 neurons Na -channel  was  and  with  induced  R-  Mg-free  media  slices  of TTX,  of  perfusion  perfusion  of  d e p o l a r i z a t i o n of  with  Mg-free  ~20 mV  in  These r e s u l t s are summarized i n T a b l e 2. gradual  l e v e l s o f hypomagnesia,  d e p l e t e d from t h e i r i n i t i a l  removal  of  [Mg  2+  ] .  The  effects  of  were s t u d i e d i n s l i c e s t h a t were g r a d u a l l y  e x t r a c e l l u l a r Mg  2+  c o n t e n t by s e q u e n t i a l  H. EL-BEHEIRY  111  0 mM Mg  15  ACh  Glu  GABA  F i g . 39 B l o c k a d e o f ACh and GABA a c t i o n s i n M g - f r e e p e r f u s a t e . (A) spontaneous depolarizing waves evoked in a sensorimotor neuron ( V = -70 mV) from a n e o c o r t i c a l slice incubated i n Mg -free media. (B) ACh and GABA r e s p o n s e s were a t t e n u a t e d i n neuron ( V = -75 mV) by removal of M g from the media. V e r t i c a l bar i n d i c a t e s 15 mV i n B. 2 +  m  m  2 +  mV  H. EL-BEHEIRY  Glu  NMDA  112  I  OmM Mg 1 JL M TTX +2  A  A  A  2 0  4 0  mV  s  F i g . 40 P o t e n t i a t i o n o f NMDA responses i n t h e M g - f r e e b a t h i n g solution containing TTX. NMDA-responses were greatly potentiated, w h i l e the G l u - e v o k e d d e p o l a r i z a t i o n s were d e p r e s s e d .  H. EL-BEHEIRY  Table 2  E f f e c t s of M g - f r e e media on s e n s o r i m o t o r  **  D e p r e s s i o n of ACh r e s p o n s e s GABA responses  7/7  P o t e n t i a t i o n of NMDA r e s p o n s e s  13/13  ***  113  neurons*  Glutamate Responses D e p r e s - No change Potension tiation 3/9  10/10  4/9  2/9  * Hyperpolarization i n7\ put resistance /V  Depression  was  (±10%)  was  observed  in  8/16  was d e t e c t e d i n 5/16  defined  as  of  the  neurons.  A  decrease  in  neruons.  >20% a t t e n u a t i o n  of  control  amplitudes  of  ACh-  o r GABA-induced d e p o l a r i z a t i o n . Potentiation  was  g l u t a m a t e o r NMDA.  defined  as  >20%  increase  of  control  amplitudes  of  H. EL-BEHEIRY  perfusion of  the  o f ACSF s o l u t i o n s  Mg-cation  depolarizations  from  are  containing  the  shown  [Mg  2+  ].  The  e x t r a c e l l u l a r environment in  r e s p o n s e s were not observed  low  F i g . 41.  A  gradual  i n o t h e r neurons  114  e f f e c t s of on  the  removal  Glu  depression  induced  of  the  (7/10) t h a t showed no change  Glu or  a s l i g h t p o t e n t i a t i o n of t h e Glu a c t i o n s . However,  the  effects  of  GABA were  consistently  reduced  by  the  gradual  2+ depletion  of  Mg  in  was slow but u s u a l l y  a  dose-dependent  manner  in  11/11  neurons.  was observed w i t h i n the e x p e r i m e n t a l p e r i o d .  Recovery A  single  an  atten-  2+ application  of  low Mg  solution  (0.5 mM)  for  15-20 min produced  u a t i o n o f t h e GABA responses t h a t was c o m p l e t e l y r e v e r s i b l e ( F i g . 4 2 A ) . On 2+ t h e o t h e r hand, i n c r e a s i n g the [Mg ] t o 3.5 mM i n the p e r f u s i n g media had no e f f e c t on t h e GABA-evoked d e p o l a r i z a t i o n s ( F i g . 42B). 2+ 6.1.3 E f f e c t s o f i n c r e a s i n g [Mg ]. Neurons (n = 8) i n s l i c e s t h a t were  incubated  spontaneous 2+ [Mg  ]  in  and  the  not  just  neurons  neurons. tions. tions  all  exhibited  a  for  During  produced  a  F i g . 43).  neurons  (8/8).  steady  A in  slight the  perfusion  increase  Applications However,  hours  perfusion  corresponding  showed  extensive  with  increasing  progressive  depolarization  input  Linder t h e s e c o n d i t i o n s t h e neurons sequential  3-7  suppression  f i r i n g and s y n a p t i c background w h i c h had been  impalement.  w i t h a change  However,  neurons;  solution  activities.  spontaneous  after  associated  ACSF  synaptic  o f t h e exaggerated present  Mg-free  the  in of Glu  resistance  Glu  was  which  observed  in  was 6/8  d i d not respond t o ACh a p p l i c a 2+  with 0.5, the  (±4 mV)  1 and  ACh-evoked evoked  responses  2 mM Mg  concentra-  depolarizations  depolarizing  responses  (5/8 in  were u n a f f e c t e d (4/8) o r 2+ i n [Mg ] (cf. F i g . 43). The p o t e n t i a t e d i n a dose-dependent  enhanced (4/8) by t h e s y s t e m a t i c i n c r e a s e GABA-induced d e p o l a r i z a t i o n s were g r a d u a l l y 2+ manner by t h e i n c r e m e n t s i n [Mg ] ( F i g . 44).  2mM Mg 1 mM Mg  0.5 mM Mg  0 mM Mg  15min  2mM Mg  20 min  20 mV 45 s m r— i CD  ,i\ij _ « r o M M 2+7 (c mM Mg ) 9  m  c  *Jr«™\.™?*\<*. ^ o .Joi mn a neuron r w. «,u . c ^u.. i C i u jr y r d u u d i removal or L^g a neurc P °very was obtained after 20 min in control solutions  C o m  l e t e  rec  3  H. EL-BEHEIRY  116  A  F i g . 42 Depression o f GABA-evoked d e p o l a r i z a t i o n by low [ M g ] . (A) p e r f u s i o n w i t h 0.5 mM M g c o n t a i n i n g media depressed the responses t o GABA i n an a n t e r i o r c i n g u l a t e neuron ( V = -68 mV). (B) i n c r e a s i n g l e v e l s of [ M g ] had no e f f e c t on GABA-induced d e p o l a r i z a t i o n s . 2 +  0  2 +  m  2 +  0  H. EL-BEHEIRY  117  Mg-free  Glu  ACh  •  20 s  •  F i g . 43 Gradual p o t e n t i a t i o n o f A C h - a c t i o n s i n a neuron ( V = -70 mV) due to a sequential i n c r e a s e of [Mg ] . Recordings were o b t a i n e d i n a s l i c e t h a t had been i n c u b a t e d i n f r e e Mg-media f o r ~3 h o u r s . V e r t i c a l bar i n d i c a t e s 15 mV. m  2 +  0  H. EL-BEHEIRY  118  2 5  30 s  F i g . 44 GABA a c t i o n s were g r a d u a l l y p o t e n t i a t e d by s e q u e n t i a l i n c r e a s e i n [Mg* J c o n c e n t r a t i o n i n the p e r f u s i n g media. Recordings were o b t a i n e d from a neuron i n a s l i c e t h a t had been i n c u b a t e d i n M g - f r e e ACSF f o r ~4 hours. 0  mV  H. EL-BEHEIRY  The magnitudes 2+ each [Mg ] were  of  the  depolarizations  averaged  in  18  evoked  neurons  by  Ach,  (Fig. 45),  Glu  The  and  119  GABA  at  in  the  changes  r e s p o n s e s t o ACh and GABA were l a r g e r and approached t h e i r r e s p e c t i v e maxima 2+ sooner w i t h i n c r e a s i n g l e v e l s of [Mg ] . However, t h e r a t e o f r i s e and 2+ magnitude o f t h e Glu r e s p o n s e s were l a r g e l y independent of t h e [Mg ] .  2+ 6.1.4  Effects  responses.  of  Because  external  of the  and  variability  b u i l d u p on the G l u r e s p o n s e s ,  in  a separate  explore the a l t e r a t i o n s i n Glu-responses environment  2+  Ca  w i t h o u t changing  the  total  Mg  the  exclusion  effects  of  2+  on  Mg  the  Glu  removal  and  s e t of e x p e r i m e n t s was d e s i g n e d by m a n i p u l a t i n g t h e d i v a l e n t cationic  concentration.  These  to  cation were 2+ Mg  2+ Ca and 2+ from t h e p e r f u s i n g media and t h e i r s u b s t i t u t i o n s w i t h 4 mM Co attenuated the G l u - i n d u c e d d e p o l a r i z a t i o n s by 38.8 ( ± 7 . 5 ) % i n 3 out o f 4 neurons 2+ 2  carried  out  in  the  presence  of  TTX.  Exclusion  of  both  +  ( F i g . 46A).  Substitution  of  Ca  with  by 15 ( ± 5 . 7 ) % i n 4 out of 4 neurons  Co  suppressed  ( F i g . 46B).  the  Glu  responses  Recovery was complete  after  15-20 min. 6.1.5 applied  Anaesthetic after  actions  perfusing  the  Isoflurane  (1.5 MAC;  GABA-evoked  depolarizations  ( F i g . 47A).  Althesin  responses  by  32.8  GABAergic  actions  4  slice  neurons) by  (75 nM;  more  35.5  Mg-free with  51.2  perfusion.  Mg-free  applications (±10)%  5 neurons)  ( ± 6 . 4 ) % and were  during  and  Anaesthetics  solution suppressed 55.4  attenuated  to  suppression  25-35 min.  the  G l u - and  (±7.2)% respectively G l u - and  (±8.5)% respectively  vulnerable  for  were  by  GABA-induced  ( F i g . 47B). the  The  anaesthetics  than t h e responses evoked by G l u . T h i s was i n c o n t r a s t t o t h e o b s e r v a t i o n s 2+ o b t a i n e d i n 2 mM Mg ( c f . S e c t i o n s 5.1 and F i g s . 34 and 3 5 ) . However, i n neurons where ACh e l i c i t e d d e p o l a r i z a t i o n s of 5-8 mV a m p l i t u d e i n z e r o mM 2+ Mg , i s o f l u r a n e c o m p l e t e l y b l o c k e d the e f f e c t s o f ACh ( c f . F i g . 4 7 A ) .  H. EL-BEHEIRY  30  120  -i  >  OH  ,  0  ,  ,  1  2 Mg+2 concentration  3 (mM)  F i g . 45 Dose-response curves showing t h e e f f e c t s of [Mg ] on t h e d e p o l a r i z a t i o n s evoked by ACh, G l u and GABA. Raw d a t a were o b t a i n e d from 18 neurons s u b j e c t e d t o d i f f e r e n t l e v e l s o f [ M g ] . 2 +  0  2 +  0  Control  Recovery  Ca&Mg-free 4mM C o +2  B  Ca-free 2mM Mg"*? 2mMCo  +2  20  mV  3 0 s  F i g . 46 E f f e c t s of changing C a - and M g - c o n c e n t r a t i o n s on glutamate a c t i o n s i n two neurons ( V = -68 mV) i n A and -79 mV i n B. Responses Glu were depressed i n Ca- and M g - f r e e s o l u t i o n s (A) more t h a n i n the absence of C a alone ( B ) . m  ro  2 +  m  3  H. EL-BEHEIRY  GABA  122  Glu  F i g . 47 A n a e s t h e t i c a c t i o n s on A C h - , G l u - and GABA-induced responses d u r i n g a p p l i c a t i o n o f M g - f r e e ACSF i n two neurons ( V = -65 mV i n A and -70 i n B). Isoflurane (IFL) and Althesin (AL) depressed GABA- more than Glu-responses. The r e s i d u a l ACh-depolarization in (A) was completely b l o c k e d by A l t h e s i n . C a l i b r a t i o n bars i n d i c a t e 25 s i n A and 60 s i n B, and 20 mV i n A and B. m  H. EL-BEHEIRY  6.2  123  Discussion A l t h o u g h m a n i f e s t a t i o n s o f h y p e r e x c i t a b i l i t y o f the CNS t h a t are a p p a r e n t  clinically  during  perioperatively  hypomagnesaemia  facilitate Hodgkin  1957;  simulated 2+ M  ]  for  Mg  the  in  surface  the a c t i v a t i o n of  alterations  C9  Ca  with  Llinas  in  the  L u t t g a u and G l i t s c h Unlike neocortical  mechanisms  perfusing  media  kept c o n s t a n t  such  (4 mM).  screening  of  the  and are  and  1980;  Mayer  active  membrane  almost  et a l .  and  depolarizations  of  have  Hodgkin  1957;  due  the  membrane  not  total any  that  can  (Frankenhauser  and  1984).  properties  entirely  that  to  McLaughlin  As  a  result,  induced changes  by in  et a l .  the the  1971;  1976).  hippocampal neurons (Mody et a l . 1987), exposure s l i c e s t o M g - f r e e media h y p e r p o l a r i z e d t h e neurons  investigations  1987;  Consequently,  inward c u r r e n t s was a v o i d e d  passive  hypomagnesaemia  the  charge  and Walton  (Frankenhauser  Q  1982; A l d r e t e  2+  c a t i o n c o n c e n t r a t i o n was  interference  requirements  In these e x p e r i m e n t s , hypomagnesia was produced  2+  divalent  anaesthetic  the underlying pathophysiological  y e t r e c e i v e d much a t t e n t i o n .  substituting  the  ( M a r t i n d a l e and Heaton 1964; Agus e t a l  Gambling e t a l . 1988),  by  modifies  decreased  their  input  comparable magnitude  resistances.  were observed  on  In  of the i n these contrast,  several  occasions  2+ during to  the  the Mg  experiments.  interference with  K-channels 1988).  -buildup  blocking  ( L l i n a s and Walton 1980; c f .  According to t h i s  sustained  a tonic  The l a t t e r action  Iseri  resulting  of  Mg  on  probably  due  Ca-activated  and F r e n c h 1984 and  scheme, t h e C a - c h a n n e l s  b l o c k a d e by M g - c a t i o n s  e f f e c t s were 2+  Begenisich  would be r e l i e v e d from t h e  i n an enhancement o f t h e  resting  C a - a c t i v a t e d K-conductance and t h e r e b y a h y p e r p o l a r i z a t i o n . 6.2.1 suppression  Suppression  of  the  of the A C h - i n d u c e d  ACh-induced responses  responses.  by removal  The dose-dependent 2+ o f Mg from t h e media  H. EL-BEHEIRY  has  not been r e p o r t e d p r e v i o u s l y  phoretic  applications  carbachoi-evoked dorsal  root  of  Mg  responses  ganglia  2+  for have  recorded  (Davies  and  neocortical  neurons.  been  to  found  Watkins  1977;  However,  depress  e x t r a c e l l u l a r l y in  the  the  spinal  and  Watkins  Evans  124  ionto-  ACh- and cord  1978).  Recent r e p o r t s d e s c r i b e a b l o c k a d e of ACh a c t i o n by a r e d u c t i o n i n [Mg in  cardiac  muscle  (Kurachi  et  al.  1986;  1988)  and  antagonize  n i c o t i n i c a c t i o n s on c e r e b e l l a r neurons  neocortex,  the  depression  of  ACh  responses  in  a  failure  the  present  a  decrease  inwardly  in  directed  K-efflux  (Wanke  depression  [Mg Ca  et  ]  would  remove  conductance,  al.  1987);  the  tonic  thereby  this  would  to  1987).  In  mechanisms:  increasing  of  an  Ca-dependent  the  of t h e i n p u t conductance and the t i m e - and  ] 2+  Mg  inhibition  decrease  2+  investigations  c o u l d be a t t r i b u t e d t o one or a c o m b i n a t i o n of the f o l l o w i n g 2+ (1)  of  (Garza e t a l .  and  ACh-induced  voltage-dependent  M-current, 2+ (2)  decreasing  [Mg  ]  may  cause  a  decrement  in  intraneuronal  2+ [Mg ] (Baker and C r a w f o r d 1972; Heinonen and Akerman 1986) thereby enhancing t h e i n w a r d l y r e c t i f y i n g K-conductance which would i n t e r f e r e w i t h the a c t i o n of ACh ( c f . K r n j e v i c ' e t a l 1967 and S t a n f i e l d 1988), 2+ (3)  low  [Mg  ]  favours  the  with high a f f i n i t y binding this  is  suggested  conversion  of  the  muscarinic  s i t e s t o low a f f i n i t y c o n f o r m a t i o n a l  by t h e observed s h i f t  to the  left  of t h e  receptor states;  occupancy-  concentration curves f o r carbachol binding i n the c e r e b r a l c o r t e x 2+ whenever Mg was added t o the i n c u b a t i n g medium (Hulme e t a l . 1980; G u r w i t z and S o k o l o v s k y 1980; B i r d s a l l e t a l . 1984; Aronstam e t a l 1985). 2+ 6.2.2  Changes  in  responses.  Previous  showed  substantial  no  [Mg  ]  do  not  significantly  i n t r a c e l l u l a r investigations changes  in  the  in  responses  rat  affect  Glu-induced  neocortical  evoked  by  Glu  slices during  H. EL-BEHEIRY  a p p l i c a t i o n of Mg-free  solutions  (Thomson e t a l . 1985;  Sutor  125  et a l .  1987).  both  direc-  2+ In  the  tions  frog  and  rat  spinal  had no a p p r e c i a b l e  cords,  e f f e c t s on t h e  G l u r e s p o n s e s (Evans e t a l . 1977; The  pooled  data  depression  of  in  the  statistically  alterations  the  [Mg  i n t r a - or  studies  depolarizations The  absence  by of  a  Glu; an  in  Q  recorded  Lacey and N i s t r i  indicated  evoked  ]  extracellularly  A u l t e t a l . 1980;  present  significant.  of  trend  this  effect  1988).  towards  was  slight  not,  could  be  however,  due  to  an  2+ inability  of  Mg  receptors  (Baudry  predominantly IV-V  of  depression  significantly  and  Lynch  1979)  w i t h Q u i s and ka  the  (Greenamyre  to  cortex  as  e t a l . 1985; of  the  (not  influence and/or  the  may  reflect  NMDA s i t e s )  suggested  by  binding  several  if  Glu  Glu  sensitive  to  c o n f i r m e d by  sites  at  layers studies  The observed  systematic  its  interactions  autoradiographic  Monaghan and Cotman 1 9 8 5 ) .  Gl u - r e s p o n s e s ,  of  trend  studies,  in  would  2+ s u g g e s t t h a t Mg  c o u l d be a charge  depolarization. ion-selective pyramidal  The decrease electrodes  neurons  supports  in  carrier contributing 2+  e x t r a c e l l u l a r Mg  during  local  activity  application  this possibility  t o the  of  Glu  Glu-evoked  recorded to  with  hippocampal  (Pumain e t a l . 1987;  1988).  In  a d d i t i o n , the l a r g e r d e p r e s s i o n of the G l u r e s p o n s e s observed i n t h e absence 2 2+ 2+ o f Mg and Ca than i n the p r e s e n c e o f Mg i n the perfusing media 2+ (cf. Results) a l s o may be i n t e r p r e t e d t h a t Mg c a t i o n s , when p r e s e n t , permeated t h r o u g h G l u - o p e r a t e d i o n o p h o r e s . 6.2.3 2 of  [Mg  the  GABA-depolarizations 2  +  2Q>  E9 m  prevention  1988;  Gyenes  GABA-induced central  of  ]  has  been  "rundown"  et a l .  consistently  a t t e n u a t e d by  a  to  be  an  GABA^-receptor  The  have  However,  suggested in  1988).  Cl~-currents  neurons.  are  the  removal  +  not wide  effects been  of  function [Mg ] 2 +  previously  variety  essential  of  on  (Stelzer  for  et a l .  postsynaptically  reported divalent  factor  for  mammalian 2+ cations (Zn ,  H. EL-BEHEIRY 2+  2  Co  ,  brate  Ni  and  species  biochemical GABA  2  +  +  Cd  )  antagonize  (Kaneko  evidence  receptors,  A  126  and  Tachibana 2+  that  it  may  responses  [Mg be  to  GABA  In  the  1986).  ]  affects  assumed  that  the the  in  different  absence  binding  of  verte-  conclusive  characteristics  dose-dependent  of  inhibition  of  t h e d e p o l a r i z a t i o n s produced by GABA a p p l i c a t i o n s  i n the p r e s e n t i n v e s t i g a 2+ tions is due t o a decrease in the i n t r a c e l l u l a r Mg concentration 2+ 2 ([Mg 3^) subsequent to the removal of [Mg ] (Baker and Crawford 1972; Heinonen and Akerman 1986; c f . F r y 1986) which l e a d s t o a "rundown" o f +  GABA^ r e c e p t o r f u n c t i o n . 2+ 6.2.4  Enhancement o f neuronal  e x c i t a b i l i t y by t h e removal of [Mg  ] .  2+ The  r e d u c t i o n of  cortical 1986;  systems  [Mg  ]  is  Q  known  to  i n c l u d i n g hippocampus  S t a n t o n e t a l . 1987;  enhance  and e n t o r h i n a l  Mody e t a l . 1987;  excitation in  cortex  as w e l l  as n e o c o r t e x (Thomson 1986;  al.  Several  mechanisms  Tancredi  A v o l i e t a l . 1987;  have been proposed  many  (Walter et a l .  Hamon e t a l . 1987;  e t a l 1990) 1989).  neuronal  f o r these  Aram e t  actions,  inclu-  ding: 2+ (1)  removal  Ca  of  entry  antagonistic  (Katz  and  and Heineman 1986; (2)  actions  Miledi  1969;  Czeh and Somjen  reduced s u r f a c e c h a r g e  of  Mg  on  Llinas  and  p r e - and Walton  (3)  removal  associated  the  ionophores  e t a l . 1986; 1987;  of  screening  and an  Mayer e t a l . 1984) voltage-dependent (Davies  and Watkins  Mody e t a l . 1987;  Mayer e t a l .  1988;).  1980;  Lambert  1989), associated  inward c u r r e n t s and a c t i o n p o t e n t i a l s (Frankenhaeuser L l i n a s and Walton 1980;  postsynaptic  Stanton  facilitation  and Hodgkin  of  1957;  and  Mg-blockade 1977;  of  Thomson  e t a l . 1987;  NMDA  receptor  1986;  MacDonald  Walter et a l .  H. EL-BEHEIRY In t h e p r e s e n t  investigations,  127  t h e p o t e n t i a t i o n of t h e NMDA r e s p o n s e s  in  2+ low  [Mg  ]  is  mechanism  that  conditions  of  GABA-ergic  consistent may  with  the  contribute  hypomagnesia  transmission;  above  to  is  the  the  thus,  the  considerations.  neuronal  6.2.5 2+ Mg  Anaesthetic-induced  -free  the  media.  These  depressant  investigations  actions  of  GABA-evoked  depolarizations  f i n d i n g was  the  responses.  Both  with  reversed  of  the  concentrations. these  agents in was  of  Glu  and  and  GABA  for  Althesin  [Mg  ] ,  insignificant of  the  relieved  of  responses  on  the  such  increased  the  An  in  time,  G l u - and interesting  transmitter-induced  amount  at  first the  p r e f e r e n t i a l l y depressed  normal  of  be  the  hypomagnesaemia.  suppression  p a r t l y e x p l a i n the  patients suffering  7  in  Though the mechanism  observations  chronic  pattern  Whereas  GABA-responses  simulated  would  under  interneurons.  demonstrate,  isoflurane in  anaesthetic  Glu-actions.  depression  depression  neurons  from t h e n e g a t i v e feedback l o o p i n h i b i t i o n by  additional  hyperexcitability  dose-dependent  pyramidal  An  GABA- compared of  similar  "selectivity" anaesthetic  attenuation anaesthetic  is  not  clear,  requirements  from c o n d i t i o n s a s s o c i a t e d w i t h hypomagnesaemia,  e.g.,  in in  alcoholism.  ANAESTHETIC-INDUCED ATTENUATION OF POSTPIKE AFTERHYPERPOLARIZATIONS MEDIATED BY Ca-ACTIVATED K-CONDUCTANCE  7.1  Results The 52  neurons  experiments  had  amplitudes  usually  selected for resting of  anaesthetic  potentials  70-75 mV, and  more ability  applications negative to  fire  in  than 5  such  this  series  -65 mV, spikes  of  spike repeti-  H. EL-BEHEIRY t i v e l y during current-pulse i n j e c t i o n s . corresponded  to layers  (mean ± SD;  65.7  IV  ± 20.5  and V of  The depths of s u c c e s s f u l  typical  cortex.  The  Mn) and r e s t i n g p o t e n t i a l s ( - 7 3 . 2  1 2 8  impalements  input  resistances mV)  ± 7 . 9  remained  s t a b l e f o r p e r i o d s of 5 0 t o 2 2 0 m i n . Depression  7.1.1  of  t h e AHPs accompanying injections  (cf.  AHPs.  The  effects  5 ( i n a few c a s e s ,  Methods) were s t u d i e d  of  anaesthetic  6 ) spikes  evoked  i n the 5 2 neurons.  application by  on  current-pulse  The c o n t r o l  AHPs  ranged from 3 . 5 - 1 1 mV i n a m p l i t u d e and 7 0 0 - 2 8 0 0 ms i n d u r a t i o n . A p p l i c a t i o n s of ible  depressions  (Figs.  isoflurane  in the These  48-50).  or A l t h e s i n  amplitudes  Methods).  and d u r a t i o n s  e f f e c t s were observed  a n a e s t h e t i c evoked changes ( u s u a l l y (cf.  produced  Isoflurane  reduced observed  MAC,  min  for  attenuation  The  ECgQ  for  approximately  of  the  after the  AHPs  in  revers-  all  DC-compensation  2 - 5 mV, i f p r e s e n t )  in resting  cases  for  any  potential  a d m i n i s t e r e d a t 1 MAC reduced the mean a m p l i t u d e (Fig. 5 0 ) .  the mean a m p l i t u d e and d u r a t i o n  by ~ 6 8 % and ~ 7 5 % , 9-15  of t h e  during  by ~ 2 3 * 4 . 8 1 % and t h e mean d u r a t i o n by ~ 2 6 ± 4 % c o n c e n t r a t i o n of 2 . 5  dose-dependent,  respectively  (Fig.  discontinuing AHP  shortening  of  AHP  recovery  application  was  7 0 LIM  was  which  150  nM.  corresponds  (Smith  Recovery  was  observed  a p p l i c a t i o n s o f 1 5 - 3 0 0 nM d o s e s . nM  dose  95.5 after  reduced  ± 18.5%,  The  mean  to control  percent  amplitude  perfusion  changes  in  1 0 to  1 2 min  after  terminating  Although A l t h e s i n a p p l i c a t i o n s at the  respectively (Fig. 5 0 ) ,  returning  panel).  the  at  brain  EC^Q  i n t r a v e n o u s dose of A l t h e s i n t h a t induces an a n a e s t h e t i c s t a t e i n v i v o 1974).  rat  The  was  single  al.,  determined i n  t h e AHPs were  application.  Althesin  durations  isoflurane  after a  et  t o the c o n c e n t r a t i o n s  by  of  Complete  50).  isoflurane  amplitude  A t an  the  and  duration  by  85.5  * 13.6%  1500 and  no r e c o v e r y was observed even a t 3 0 min at a rate of amplitude  and  s i g n i f i c a n t s t a t i s t i c a l l y a t a l l doses of both agents  3 ml/min  (Fig.  49,  last  of  AHPs  were  duration (Fig.  50).  H. EL-BEHEIRY  129  F i g . 48 I s o f l u r a n e induced an a t t e n u a t i o n of t h e a f t e r h y p e r p o l a r i z a t i o n s (AHPs). I s o f l u r a n e a p p l i c a t i o n s t o t h r e e neurons i n 0 . 5 , 1.5, 2.5 MAC doses produced dose-dependent and r e v e r s i b l e r e d u c t i o n s i n t h e d u r a t i o n and ampli t u d e of the AHPs.  controls  Isoflurane 0.5MAC  recoveries  controls  Althesin  recoveries  15x10" M 6  F i g . 49 Depression of the afterhyperpolarizations (AHPs) by a p p l i c a t i o n s of A l t h e s i n . The e f f e c t s were evident at d i f f e r e n t doses (within two log units of alphaxalone content). The depression by the highest dose of A l t h e s i n was i r r e v e r s i b l e even a f t e r 30 min in control s o l u t i o n .  F i g . 50 Dose-response r e l a t i o n s h i p s f o r the d e p r e s s i o n of the a f t e r h y p e r p o l a r i z a t i o n s produced by a p p l i c a t i o n s of i s o f l u r a n e (6-9 min; A,C) and A l t h e s i n (4-6 min; B,D). The d a t a p o i n t s were f i t t e d by 3rd o r d e r r e g r e s s i o n a n a l y s i s . Symbols ( • ) i n d i c a t e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s from the mean c o n t r o l values at p < 0.05.  H. EL-BEHEIRY  7.1.2  to  B i c u c u l l i n e - b l o c k a d e of i n h i b i t o r y p o s t s y n a p t i c  potentials  B i c u c u l l i n e a p p l i c a t i o n s were used i n 8 n o n - s p o n t a n e o u s l y  active  reduce  DC  generated (cf.  "contamination"  could  through  the  through Cl-conductances  McCormick  IPSPs  of  and  have  occurred  recurrent  released  GABA  Prince  the  bicuculline  was  epicortical  electrical  by  synaptic  mediated by  receptors  1986).  For  if  current-pulse  the  collaterals,  onto  AHPs  a  synaptically  recorded  chosen  because  (cf.  Althesin  (100  uM) were chosen  clinical  r e l e v a n c e (Smith e t a l . 1974;  Bicuculline facilitated small  increase  were  approximately bicuculline  7.2  {~10%) i n  Rj  The  recovery  had  with  activated, that  concentration  of  IPSPs  by  blocked  evoked  in  isoflurane t i m e s as  AHPs  such  (1.5  well  slice  MAC)  as  for  affect  V  and their  C u l l e n 1986).  alone  was  of  of  interneuron  that  completely  doses  for fast  bursts  50 uM  potentials  endogenous GABA  injections  A  neurons  did  not  evoked  greatly  on c u r r e n t - p u l s e  observed  during  and  m  injections.  bicuculline  much  ( F i g . 51). the  same  ( F i g . 52).  more The time The  than  with  the  sole  anaesthetic courses changes  in in  administration  actions the V  absence  and  m  on  R.  the and  induced  i n t h e p r e s e n c e o f b i c u c u l l i n e t h a n i n i t s absence  A  application.  o r A l t h e s i n was a p p l i e d c o n c o m i t a n t l y w i t h b i c u c u l l i n e ,  reduced  anaesthetic  were l e s s  application  r e p e t i t i v e spike  When i s o f l u r a n e AHPs  2).  for  summation  observations  were  preparations  a  and  connected  neuron.  of  stimulation  Section  example,  133  of  the the  AHPs  had  presence  of  by  (Fig.  Althesin 52B).  Discussion The most  striking  of i n v e s t i g a t i o n s well spike  as  the  were t h e dose-dependent  shorter  discharges.  e f f e c t s of both agents  durations These  of  the  postsynaptic  on a l l  neurons  attenuations  AHPs  associated  effects  could  in  this  series  in the amplitudes, with not  be  the  as  repetitive  attributed  to  H. EL-BEHEIRY  134  F i g . 51 Isoflurane (IFL; 1.5 MAC f o r 6 min) and A l t h e s i n (AL; 100 nM f o r 4 min) i n d u c e d d e p r e s s i o n s of the a f t e r h y p e r p o l a r i z a t i o n s (AHPs) i n two neurons (A,B) where bicuculline methiodide (BIC; 50 nM) had been additionally applied. The AHPs were a t t e n u a t e d more i n the presence of b i c u c u l l i n e t h a n i n i t s absence. Note t h a t a d e p r e s s i o n o f t h e AHPs o c c u r r e d w i t h the a p p l i c a t i o n of b i c u c u l l i n e a l o n e .  F i g . 52 The mean changes i n r e s t i n g membrane p o t e n t i a l , i n p u t r e s i s t a n c e , and a f t e r h y p e r p o l a r i z a t i o n (AHP) a m p l i t u d e or d u r a t i o n i n e i g h t neurons were induced by i s o f l u r a n e (A; I F L , 1.5 MAC f o r 6-9 min, n = 4) and A l t h e s i n (B; A L , 100 uM f o r 4-6 min, n = 4 ) . Note t h e pronounced e f f e c t s of IFL on the AHP i n the presence of b i c u c u l l i n e (BIC) i n A. (B) b i c u c u l l i n e p a r t i a l l y b l o c k e d the changes i n r e s t i n g p o t e n t i a l and i n p u t r e s i s t a n c e , and p o t e n t i a t e d t h e d e p r e s s i o n of the AHP induced by A l t h e s i n .  H. EL-BEHEIRY  anaesthetic  induced  been observed cells  changes  during  in  similar  resting  membrane  applications  ( F u j i w a r a e t a l 1988; Miu and P u i l  neurons  ( P u i l e t a l . 1988).  AHPs has tics,  been demonstrated  and  barbiturates  (Bosnjak  isoflurane  1989)  A depression during  of  al.  to  and  of d i r e c t l y o r of  1982;  and  was  anaesthetics Prince as  the  1986).  with  blockade due Both  to  activity. of  AHPs,  In  of  Ca-activated  had  blockade  applied.  It  such c a s e s ,  is  unlikely  the  McCormick  and  IPSPs w i t h t h e AHPs as  have  been  potentiated  e t a l . 1987)  application  c o u l d be  alone  by  Therefore,  a t t r i b u t e d mainly  to  g e n e r a t i n g t h e AHPs.  the  the  prevented  by  in  of  more  an  GABA-ergic pronounced  bicuculline, (cf.  Schwindt  neurons that  a  occlusion  the  activity effects  suggesting et  of  the  al.  some  1988a,  been  the  avoided.  AHPs  occlusion 1988b)  where t h e G A B A ^ - a n t a g o n i s t non-Ca-dependent  greater  AHP-conductances  had on  a  by had  K-conductance  effects  anaesthetic  observed actions  in  these  directly  investigations on  the  ionic  under of  the  a GABAnot  been  interfered  w i t h , o r c o n t r i b u t e d t o , t h e g e n e s i s of the AHPs ( c f . Schwindt e t a l . 1988b).  well  decreased  A l t h e s i n a p p l i c a t i o n produced  because  much  K-conductance  mediated C I - c o n d u c t a n c e  (Barker  with  o f the AHPs s u g g e s t i n g a b l o c k a d e of the endogenous  potentiation  anaesthetics  may  Bicuculline  presumably  anaesthetic  conditions  by A l t h e s i n  neurons.  a m p l i t u d e s and d u r a t i o n s  that  of  (cf.  1985;  In some o f  concomitantly  CI-conductances  summations  GABA-actions  especially  neocortical  GABAergic  GABA-mediated  Hence, t h e l i k e l y  endogenous  anaesthetic, in  to block  administered  anaesthe-  Macdonald  the  bicuculline  ganglion  volatile  1989; Southan and Wann 1989).  studies,  CAj  synaptically-evoked  other  Werz  have  hippocampal  F u j i w a r a e t a l . 1988; Miu and P u i l present  also  and human s y m p a t h e t i c  applications et  properties  136  1988a,  could  be  mechanisms  H. EL-BEHEIRY  7.2.1  Mechanism(s)  Normally,  the AHP i s  depolarization certain  [Ca  ].  anaesthetic  a consequence  and  Ca-entry.  K-channels  K-current. 2+  of  (cf.  As  that  a K-conductance a  K r n j e v i c ' et  inactivates  the  Ca-sequestration.  intracellular  energy  ( S m i t h e t a l . 1974; interference neocortical  result, al.  with  Halothane,  stores Morris  the  neurons  thereby 1986;  be  7.2.2  t h a t was  related  and  generation.  is  i n i t i a t e d by  rises,  initiates  of  Gustafsson  the  1974;  neocortical regulating  neuronal  sedatives  increased  subcortical  Althesin  of  slow  K-conductance by  activates an  neocortex,  a  anaesthetic  blockade  outward  AHPs  inhibition in  al.  (Schwindt  et  AHPs  The  al.  depression  not  1988).  1978b) al.  and  blockade  of  1988), of  reduced  and  Carlen  al.  of  the  Morris  repetitive firing AHP.  of  also  1988),  spike  or  in  the  The and  AHPs  in  appreciated observed  firing  ability  apamin  could  Puil  in  generation  The  in  1986).  for that  various in  such  neurons  by  discharge.  In  accompanied  the  apparent  t h a t w e l l - d e v e l o p e d , f a s t AHPs i n  applications  AHP  neurons.  (1985)  AHP  the  (Baldiserra  well  repetitive slow  deplete  the  o r i n aged hippocampal  increases repetitive  et  on  mechanisms  medium-duration is  t h e medium-duration  by  the  and  alterations  neocortical  1988a,b)  reduced  of  or A l t h e s i n  neurons  of  Therefore,  (Krnjevic'and  subcortical  hyperexcitability. the  in  1984)  barbiturates  mechanism  inward C a - c u r r e n t s  AHP  be r e l a t e d t o t h e o b s e r v a t i o n s are  Chad  Ca-buffering  isoflurane  neurons by apamin (Zang and K r n j e v i c ' 1987) et  and  compromising  produced  et  neurons.  (Niesen  and  less specific anaesthetic 2+ / o f [Ca ] . ( c f . K r n j e v i c 1974a;  Krnjevic  neurons  (Eckert  to  Significance  importance  neurons  ].  D a n i e l 1 and H a r r i s  Ca-activated  s t e a d y - s t a t e background  ethanol  AHP  that  [Ca  1978b)  Ca-channels  be a d i r e c t r e s u l t of d e c r e a s e d may  with  The decay o f t h i s c u r r e n t i s p a r t l y c o n t i n g e n t on the b u i l d u p  internal  or  of  interference  137  ethanol  paradox  may  hippocampal (Zhang  and  H. EL-BEHEIRY  Krnjevic  1987;  and  are  not  The  anaesthetic  consequence  Niesen  prominent  of  e t a l . 1988). in  neoocortical  depression a removal  primed f o r a c t i v a t i o n .  Such AHPs a r e  of  of  neurons  repetitive  the major  138  not m e d i a t e d by  (Schwindt  et  al.  2 +  1988a,b).  firing  in  neocortex  mechanism  by  which Na-channels  F o r example, d e p r e s s i o n  Ca  could  be  a  are  of the medium-duration  AHPs  i n n e o c o r t e x by a n a e s t h e s i a would p r e v e n t the l a t e r e p o l a r i z i n g s h i f t o f the membrane p o t e n t i a l which excitation  cycle  for  r e p e t i t i v e spike  8  normally  decreases  Na-activation,  N a - i n a c t i v a t i o n and  resulting  in  an  resets  the  attenuation  of  discharge.  EFFECTS OF ISOFLURANE ON THE GLUTAMATE- AND POTASSIUM-INDUCED INCREASE IN INTRANEURONAL CALCIUM CONCENTRATION  The o b j e c t i v e s of t h i s s e r i e s of i n v e s t i g a t i o n s  were:  (1) t o e l u c i d a t e t h e mechanisms of t h e d e p r e s s a n t a c t i o n s o f on the ACh- and G l u - e v o k e d d e p o l a r i z a t i o n ( c f . S e c t i o n  anaesthetics  5), 2+  (2)  to  monitor  possible  changes  in  the  resting  level  of  [Ca  ].  i n d u c e d by i s o f l u r a n e , and (3)  to  study  the  effects  of  isoflurane  a c t i v a t i o n o f v o l t a g e dependent Cultured fetal partly  because  hippocampal  they  on  the  p r e - and  postsynaptic  Ca-channels.  neurons  represent a well  were chosen  for  these  c h a r a c t e r i z e d model  investigations  for  the  study  of  2+ the  Ca  homeostasis  in  the  central  nervous  system  (cf.  Kudo  and  Ogura  1986). 8.1  Results Resting  [Ca *]^ 2  levels  were  63.8  (± 38.9)  nM  (n = 3 0 ) .  were measured f o r ~2 min p r i o r t o a g o n i s t o r d r u g a p p l i c a t i o n ;  [Ca  neurons  2 +  ]  1  with  H. EL-BEHEIRY resting that  [Ca *]..  values  2  f l u c t u a t e d more  above  than  150 nM  ±20 nM were  or not  those  with  resting  included  for  analysis.  c o n d i t i o n s o c c u r r e d r a r e l y and seemed t o be r e l a t e d to poor c e l l Similar  volumes  of c o n t r o l  139 [Ca  2 +  ].  [These  viability.]  s o l u t i o n were i n j e c t e d on d i f f e r e n t o c c a s i o n s  to  t e s t f o r i n j e c t i o n ( p o s s i b l y mechanical) a r t i f a c t s . 8.1.1 2+ [Ca  Response  ].  were  micromolar  to  ACh  observed  range  and  (3/12  carbachol. neurons)  (10-200 nM)  was  Small  when  applied  ACh  in  i n j e c t i o n as a bolus  i n t o t h e r e c o r d i n g chamber.  latency  a duration  of  ~20s  observations brain  and  of  Benavides  100-150s.  and a s s o c i a t e s  synaptoneurosomes.  The  because  agonists  were too small i n t h i s model.  Continuous  Effects  of  perfusion  produced o n l y small a p e r i o d o f 20 m i n . evoked  a  increments  mean  instantaneous  in  isoflurane  of  media  of  on  ±78  and had an " o n / o f f "  added t o the p e r f u s i n g  solution,  volumes  is  cf.  ].  with  nM  in  type of  nM) in  Kudo  by  [Ca *].j  the  the  by  had a  with  the  1988) be  in well  cholinergic  Glu  actions.  isoflurane 2+ i n t h e r e s t i n g [Ca ] . o v e r (25 Ltl volumes) t o 18 neurons 2+ [Ca  pattern  s i m i l a r responses  and  not  and  2  1.5  et a l .  could  in  (25 ul)  consistent  evoked  resting  saturated  220  carbachol  of i s o f l u r a n e 2+ [Ca  (~25  The evoked responses This  and i n c o n s i s t e n t changes A p p l i c a t i o n of 50 LIM Glu  increase  or  small  (1988;  effects  studied  8.1.2  the  of  increases  ].. (Fig.  2.5  MAC  The  responses  53).  were  When TTX  was  were o b t a i n e d w i t h 50 uM  Glu a p p l i c a t i o n . 2+ Perfusion  of  isoflurane  increases  evoked  by  ( F i g . 53)  in  free  favouring  NMDA  2.5 MAC  TTX or  isoflurane  Glu  Quis  (4/8  media. was  (Figs.  0.5  neurons)  had  and  no  or  Attenuation always  54  MAC  potentiated of  observed 55,  effects  the on  Glu  on the  Glu  responses  application  Table 3).  the  [Ca  ].  responses in  media  of  1.75  and  Isoflurane  2.5  MAC  H. EL-BEHEIRY  140  F i g . 53 P o t e n t i a t i o n o f r e s p o n s e s t o g l u t a m a t e i n j e c t i o n s (25 uM; • ) i n 5 c o n s e c u t i v e l y sampled neurons by 0.5 MAC i s o f l u r a n e under QUIS c o n d i t i o n s .  H. EL-BEHEIRY  141  300 •  IFL 1.75 MAC  200 dD CM  +CO  o  — 100  3  ftCbg  *-qJ  \  b-^  a  0  h  0  20  40  60  Time Imin)  Fig. 54 Isoflurane (1.75 MAC) inhibition of increases in produced by glutamate application (50 uM; A ) under NMDA conditions.  [Ca ]j 2 +  H. EL-BEHEIRY  142  600.  A  H 0  A  A 1  A  A 1  1  20  A 1  A  A  A 1  40  1  A  A  A  r-  1  60  80  Time (min 1  Fig. 55 Isoflurane (2.5 MAC) blockade of increase g l u t a m a t e a p p l i c a t i o n (50 M ; • ) under Quis c o n d i t i o n s . U  in  [Ca ]i 2 +  by  H. EL-BEHEIRY Table and  3  Effects  of  isoflurane  on  [Ca  ].  increases  evoked  by  143 glutamate  K . +  Increases i n [Ca ]-{  Depression*by  2+  evoked by NMDA  {TTX  0.5  isoflurane 1.5  (MAC)  1.75  2.5  64 ± 14.4% (n = 4)  present)  (No TTX)  No e f f e c t * * (n = 3)  60 * 10% (n = 8)  QUIS (TTX  K  present)  52 ± 12.4% (n = 8)  (No TTX)  41 ± 15.3% (n = 4)  (TTX  45 ± 21% (n = 6)  85 * 13.4% (n = 4)  +  *  present)  Depression  glutamate or K  was +  defined  as  >20% decrease  i n j e c t i o n s than i n c o n t r o l  in  the  responses  conditions.  P o t e n t i a t i o n o f g l u t a m a t e responses was observed i n 5  neurons.  induced  by  H. EL-BEHEIRY completely  suppressed  t h e Glu responses  such t h a t o n l y p a r t i a l  o b s e r v e d w i t h i n the e x p e r i m e n t a l p e r i o d consistent  d e s p i t e the presence  responses  or  (Fig.  absence  r e q u i r e d 6 min of a n a e s t h e t i c  55).  of  These  TTX.  recovery  was  observations  Inhibition  application.  144  Recovery  of was  were  the  Glu  observed  a f t e r 8-12 m i n . 8.1.3 (50 mM)  Isoflurane slowly  increased  [Ca  56).  the  In  actions  injected  2 +  ]  by  i  as  300  presence  a  of  Isoflurane 2+ [Ca ].j evoked  + K  TTX.  Similar  blocker in 4 8.2  by  (n = 15)  the  recording  increments  injections  were  into in  administration  bolus  in  in  the of  absence 2+ [Ca  ].  depressed  by  observed  the  45 * 22% the  [Ca  of  presence  chamber (Fig.  slightly  increase  the of  K  TTX  were  the in  ]..  in  absence  the  of  Na-channel  neurons.  failure  of  isoflurane  consistently  increase  observations  in  synaptosomes  (Daniel1  rat  the d i s c r e p a n t  cultured  neurons,  basal  and  higher  levels  of  cell  of  is  their  poorer  [Ca  ].  not  high in  doses  (Morris  and  with  1986)  to  other  and  used may  o f synaptoneurosomes, stability  ( 2 . 5 MAC)  agreement  D i f f e r e n t models  survival  as r e c e p t o r s . 2+  at  suspensions  1988).  brain account  compared  probable  with  hetero-  In a d d i t i o n they p o s s e s s s i g n i f i c a n t l y than  those  reported  for  intact  cells  1988).  In the p r e s e n t i n v e s t i g a t i o n s , absence  ].  A drawback  resting  ( c f . Benavides et a l .  [Ca  Harris  results. is  application 2+  thymocyte  g e n e i t y i n s i z e as w e l l  or  bolus  increases  Discussion The  for  MAC)  depressions  nM  TTX  (Table 3 ) .  K-evoked  25 u l  ±95  g r e a t e r i n 7 neurons (1.5  on  800 uM Mg  in  the  G l u responses bathing  were evoked i n t h e  solution.  This  presence  concentration  s u f f i c i e n t f o r b l o c k a d e o f t h e NMDA-type o f r e c e p t o r s a c t i v a t e d by G l u  is  (Nowak  H. EL-BEHEIRY  145  Fig. 56 Voltage-gated increase in [Ca ]i produced by K (50 and 100 LIM, • ) b o l u s i n j e c t i o n s t h a t were depressed by i s o f l u r a n e a d m i n i s t r a t i o n under Q u i s c o n d i t i o n s . The a n a e s t h e t i c d e p r e s s i o n was o b s e r v e d i n the presence o f TTX. Recovery was observed w i t h t h e h i g h e s t b o l u s o f K (100 n M ) . 2 +  +  +  H. EL-BEHEIRY  et  al.  1984).  activation 1987).  Consequently,  The a d d i t i o n of TTX would have  ].  (Puil  has and  synaptic  been  the  inputs.  reported  Baimbridge  anaesthetics ting  responses  1989).  of  However,  neurons  1989.).  A transient  brief  2+ of  increase  response.  to al.  an  isolation  of t h e  neurons  the G l u - i n d u c e d as  well of  two  as  rise  G l u , by  distinct  in  halothane  interference  understood.  induces  (Murphy  mainly  (Murphy e t  mechanisms  are not w e l l  due  receptors  isoflurane  the  receptors,  hippocampal  more m a i n t a i n e d p l a t e a u  for  were  of  An a t t e n u a t i o n o f  w i t h t h e Glu a c t i o n s  Quis-subtype  ensured  previously  in vitro  removal  recorded  of e i t h e r the NMDA o r the Q u i s - t y p e  from i m p i n g i n g 2+ [Ca  the  146  of  activa-  responses  in  and  M i l l e r 1989; c f . Furuya e t a l 2+ [Ca ] . superimposed on top of a  in  The p r o l o n g e d  response  was  abolished  by  +  Ca  or  Na  sensitive  Ca-channels  dependent  Ca-channels  from  the  external  media  or  by  blocking  voltage-  (Murphy and M i l l e r 1989). In c o n t r a s t , none of t h e s e 2+ manipulations abolished the t r a n s i e n t [Ca ] . spike. Accordingly, the p r o l o n g e d response has been suggested t o r e s u l t from a c t i v a t e d v o l t a g e 2+ whereas  the  transient  [Ca  ].  spike  resulted  from  2+ Ca  mobilization  agonist-induced Nicoletti  et  receptor/G The  actions  likely  Shaughnessy  intracellular  stores  triphosphate  (IP^)  inositol al.  protein of  involve  from  1988), level  mediated (Katada  isoflurane blockade  1988;  on  of  the  al.  1985;  Glu-evoked  1988)  in  turn  synthesis  phospholipase  voltage-dependent  K r n j e v i c ' and P u i l  t h e c o u p l i n g of i o n i c c h a n n e l s receptors.  et  by  which  due  (Berridge C  effects  El-Fakahany increases Ca-channels  and/or  is  direct  et  in  (cf.  1987; at  al. 2+  [Ca  to  the  1988). ].  most  Lodge  and  interference  with  t r i g g e r e d by t h e i n t e r a c t i o n s of Glu w i t h  its  2+ + Increments of [Ca ] . evoked by K a p p l i c a t i o n s were depressed by i s o f l u r a n e d e s p i t e the presence o r absence of TTX. T h i s p r o v i d e s presumptive  H. EL-BEHEIRY evidence t h a t anaesthetics postsynaptic Section  4)  sites.  Hence,  could r e s u l t  of p o s t s y n a p t i c  a f f e c t voltage-dependent the observed  from d e c r e a s e d  These i n v e s t i g a t i o n s mainly  The  on  the  about  to  the  aspects  be  isoflurane  sites  in v i t r o  slice  metabolism  of  target  carried  in vitro  of  out,  optimal  arousal  drug  For  or  certain  be  extracortical mostly had  Because  afferent  employed  connections.  In  to  i o n i c environment  and  for  simulations  medium  (cf.  (in vivo  the  attenuation  the  in vitro  neocortical  the  anaesthetic  reservations quantitative  in  the  the  various  a s s u r e d the  pharmacokinetic  obtained may  e s p e c i a l l y f o r dose-response  studies.  of  parameters  from s t u d i e s  in  the  be m i n i m i z e d  in  the  are d e v o i d o f i n f l u e n c e s actions  are  from  attributable  p a r t i c u l a r l y when TTX the  application  emanating  from  situation  allowed manipulation  pathophysiological  conditions  of drug c o n c e n t r a t i o n s  presence  vivo  achievement  intracortical  f o r t h e e x p l o r a t i o n of t h e mechanisms of of  in  of t h e t o n i c i n f l u e n c e o f  anaesthetic  activity  in  and  that  about  the  observa-  allowed w e l l - c o n t r o l l e d  from  preparation)  investigation,  Chemical d e t e r m i n a t i o n s Methods)  results  neocortical slices  reduce  addition*  the  magnesaemia).  some of  of  qualitative  uncertainties  activity,  t o the neuron under  been  and  generalized  1981), removal  example,  animal  e.g.,  different  i n t e r p r e t a t i o n of  decerebrate  s l i c e preparation.  and  the p r o d u c t i o n  despite  may  pathways  t h a t c o m p l i c a t e the intact  release  Althesin  preparation  preparation,  that  levels.  in  and  slice  s i t u a t i o n ( c f . K o v a c h i c h and M i s h r a ascending  transmitter  (cf.  GENERAL DISCUSSION  effects  neocortical  experiments  of EPSPs and IPSPs  p r o v i d e , f o r the f i r s t t i m e , comprehensive  neurons w h i c h are l i k e l y state.  depression  at p r e - and  s e n s i t i v i t i e s to transmitters.  9  tions  Ca-channels  147  of  optimal  levels  of  drug-actions (e.g.,  in the in  the  hypobathing slice,  H. EL-BEHEIRY  9.1  N e o c o r t i c a l u n i t a c t i v i t y and g e n e r a l Substantial  spontaneous  evidence  activity  l o c a t i o n and animal  has  of  been  neocortical  species  Foster  detect  e t a l . 1982).  any  differences  anaesthesia  by  many  neurons,  depending  (Evarts  Moreover,  between  the  1964;  associational  on  Noda and Adey  Noda and Adey effects  of  that  their  the exact  cortex.  Such  modulation  1970; Webb  (1973) were  thiopental  normal s l e e p on the temporal p a t t e r n of d i s c h a r g e cat  investigators  i n v e s t i g a t e d , changes w i t h time and a c c o r d i n g  t h e a n i m a l ' s degree o f a l e r t n e s s a,b;  provided  148  of  not  to 1976  able  to  anaesthesia  from s i n g l e neocortical  neurons spike  and  i n the  activity  d u r i n g a n a e s t h e s i a has been a t t r i b u t e d t o a n a e s t h e t i c a c t i o n s on t h e a f f e r e n t pathways  (Mountcastle  Greenbaum  and  anaesthetics  units  in  preparations  spontaneous neurons. neurons  and  cortical  is  the  spikes  in  perception  of  Anaesthetic  The  of  on  the  traces  internal  actions:  would and  that  depression  be  the  reduced  external  of  firing of  of  cerebral  the  cortical  i n a b i l i t i e s of  the  the g e n e r a t i o n spread  of c e r e b r a l c o r t i c a l  (unconscious)  depression  reported  impede a t r a n s c o r t i c a l  the p r e v a i l i n g l e v e l  memory  1965;  investigations  a c t i v i t y and t h e  and g e n e r a l l y  Hance  spontaneously  present  actions  and  (1967)  anaesthetic-induced  direct  obtunded, r e s u l t i n g i n an a n a e s t h e t i c 9.2  Berry  r e p e t i t i v e l y would tend t o d i s r u p t  Accordingly, of  activities  spontaneous  patterns  generation  to  1965; Robson  animals.  that  due  Merlis  However,  attenuated  revealed  spiking  information.  1957;  1965).  The r e d u c t i o n to f i r e  al.  decerebrate  activity  meaningful  tion  Merlis  uniformly  neocortical slice  et  to the  of  activa-  extent  environments  of  that  would  be  potentiation  of  state.  excitation  or  inhibition? The  observed  selective  t r a n s m i t t e r s and c o r t i c a l  interference  EPSPs observed  with  the  actions  in the present  of  studies  activating support  the  H. EL-BEHEIRY concept  that  depression  anaesthetic  of  actions,  excitation  and  Enhancement o r p r o l o n g a t i o n mechanism  of  action  of  not  intrinsic  direct  and  certain  of  neurons  in  result  soporofic  the  of  neocortex,  potentiation  control  agents,  regulation  of  of  e.g.,  receive sustained  of  due  to  inhibition. is a  likely  benzodiazepines  or  On t h e o t h e r hand, t h e  excitatory  neuronal  are  in the b r a i n  ( C a r l e n e t a l . 1985).  extrinsic  stringent  Neocortical inputs  and  a  least  of i n h i b i t o r y p r o c e s s e s  b a r b i t u r a t e s i n s e d a t i v e doses strict  at  149  processes  excitability  provides  in  the  a c t i v a t i o n from i n t r a c o r t i c a l  a  brain. synaptic  ( c f . S e c t i o n 3) as w e l l as from a s c e n d i n g pathways, p a r t i c u l a r l y t h o s e  the  RAS  (Matsumura  particularly  in  anaesthetic  the  state.  l i p i d hypothesis lipids  of  results,  the  et  al.  neocortex This  is  plasma  however,  accompany  provides not  membrane  easy  which  are c o n s i s t e n t  depression  neocortical  Hence,  neurons induction  of  (Franks  the  may of  with  an  attenuation  a plausible to  of  excitation  c e l l u l a r basis  reconcile  that predicts a dissolution  w i t h hydrophobic p r o t e i n s The  1988).  with  the  for  non-specific  of a n a e s t h e t i c m o l e c u l e s  produces specific  anaesthesia. interactions  in the  The of  present  anaesthetics  and L i e b , 1987).  inhibitory  underlie  the  anaesthesia  responses excitatory  with A l t h e s i n  to  GABA  demonstrated  phenomena or  that  isoflurane;  9.3 The dependent  a  logical  of c o n s c i o u s n e s s  processes  in  a  high  generating degree  1965; Webb 1983) w h i c h i s l i b e r a t e d o n t o t h e neurons ascending  RAS  (Szerb  agent  ( V i c k e r s e t a l . 1981; R i c h t e r 1981).  The a n a e s t h e t i c s t a t e and s u p p r e s s i o n  on  in  reportedly  p r e v e n t i v e measure would be the p r e m e d i c a t i o n w i t h a G A B A - p o t e n t i a t i n g such as a b e n z o d i a z e p i n e  the  1967;  consciousness of  spontaneous  and  the  mechanisms  cerebral  repetitive  cortex  activity  promoted by the a c t i o n s o f ACh (Woody e t a l . as a consequence Krnjevic  1969;  of t h e s u s t a i n e d Shute  and L e w i s  are  (Libet 1978)  a c t i v i t y of t h e 1963;  Kimura  et  H. EL-BEHEIRY  al.  1981).  tion et  ACh m a r k e d l y  and s y n a p t i c a l l y  al.  1981).  The  enhances  cholinergic enhances fast,  f a c i l i t a t o r y action  (Krnjevic  neurons  t h e neuronal  in  a greater  1981,  the  of  conscious  ACh  the o v e r a l l  the  al.  level  Benjamin  are  decrease  in  conscious  the  system  background to  complete  suppression  cortical  perception  activities 1988). level  and  neocortical  of of  result  Anaesthetics of  lower  neurons  the  in  cortical  of  an  modalities  unconscious this  the  tone  RAS  or  t o ACh s y n a p t i c a l l y  and  cholinergic  either by  by  between  essential  can  "tune"  would  from  blocking the  the  Hence,  a  obtund  subcortical  Francesconi  attenuating  for  consciousness.  drive  (cf.  the  a c t i v i t y and  arousal.  arising  state  discharge  1988)  probably  activity  alertness  ascending sensory  depress of  of  degrees  the  excitability  level  is  l i k e Glu f o r a  the maintenance of a l e r t n e s s and c o n t r i b u t e t o t h e c o n t e n t o f A  to  Consequently,  transmitters  actions  receptors  of c o r t i c a l  and  stimula-  1971b; Dodd  neurons  1983).  (Puil  These  et  muscarinic  of  to "on/off"  information  state.  at  McLennan  responsiveness  transfer  of  to e l e c t r i c a l  (Krnjevic  tendency  1989;  RAS t o n i c a l l y r a i s e s  specific  responses  mediated d e p o l a r i z a t i o n  slow t o d e v e l o p and c o n f e r s repetitively  cortical  150  the  et  al.  intrinsic  responses  r e l e a s e d by t h e c o n t i n u o u s  of  excitant  a c t i o n s of t h e r e t i c u l a r f o r m a t i o n . The  results  and o t h e r s  of  French et a l .  (Haugen and M e l z a c k  (1953; c f .  1957;  that anaesthetics  i n t e r f e r e w i t h the  of  to the  sensory  inputs  due t o the g r e a t e r  cortex.  number of  also  Morruzi  D a v i s e t a l . 1957)  synaptic  findings  were  connections  T h i s t a c i t assumption  a c c e p t e d view of t h e e s s e n t i a l  central  actions  in  the  processing  i n t e r p r e t e d as  t h a t , according  (1947), would r e n d e r t h e RAS more v u l n e r a b l e t o a n a e s t h e t i c o t h e r a r e a s of the b r a i n .  1949)  f a v o u r e d t h e concept  r e t i c u l a r formation The  and Magoun  to  being Barany  a c t i o n s than  any  has remained t h e most w i d e l y of a n a e s t h e t i c s ,  despite  the  H. EL-BEHEIRY  relative  paucity  of c r i t i c a l  evidence.  Several  v o l a t i l e and i n t r a v e n o u s agents e q u a l l y depress pathways  i n d i f f e r e n t areas  1963; De Jong t h e RAS i s  e t a l . 1968).  not a p r i m a r y  a g e n e r a l i z e d or observed ( c f .  neuraxis  have  p o l y - as w e l l as (Austin  and  Recent b i o c h e m i c a l f i n d i n g s  depression  of  reported  monosynaptic  Pask also  1952;  Esplin  indicate that  a c t i o n of a n a e s t h e t i c s ,  the c e r e b r a l c o r t e x  that  is  whereas  consistently  Introduction).  that  neocortical  the  s i t e of d e p r e s s a n t  diffuse  The major h y p o t h e s i s suggests  of  papers  151  developed d u r i n g t h e c o u r s e o f these  general  depress  chemical  sensitivities  of  to s y n a p t i c a l l y released a c t i v a t i n g t r a n s m i t t e r s  thereby  a t t e n u a t i n g t h e s u s t a i n e d tone of t h e RAS and r e - s e t t i n g t h e c o r t i c a l  arousal  system  to  neurons  anaesthetics  investigations  a  anaesthetic the  lower state".  dose-response  selectively tions  steady  level  of  studies the  which  showed  in anaesthetic  i n d u c e d by G l u produced than  the  dose-dependent  of  anaesthetic  slopes the  which  the  by  situation,  achieved and  in  Althesin  extracellular applica-  corresponding  to  those  observed  in  the  dose-response  dose-response  curves  curves  for  levels  By a p p l y i n g of  both  characterizing  i n t e r f e r e n c e w i t h the e x c i t a t o r y a c t i o n s  surgical  "the  The a t t e n u a t i o n of t h e f a s t responses  degrees of a n a e s t h e t i c e f f i c a c y .  in vivo  was  isoflurane  Hence, both ACh- and G l u - e v o k e d responses a r e d e p r e s s e d varying  constitutes  hypothesis  both  evoked  concentrations  lower  slopes  that  depolarizations  in the b r a i n of anaesthetized animals.  agents  activity  P a r t i a l v e r i f i c a t i o n of t h e  depressed  o f ACh  state  of  the ACh.  a t a g i v e n dose w i t h  t h i s i n t e r p r e t a t i o n to  anaesthesia  would  be  expressed  when the t o n i c e x c i t a t i o n produced by ACh i n the c e r e b r a l c o r t e x i s  depressed  whereas t h e f a s t e r a c t i o n s of Glu are o n l y s l i g h t l y a f f e c t e d .  Deeper  levels  of the a n a e s t h e t i c s t a t e would r e f l e c t i n t e r f e r e n c e w i t h the a c t i o n s o f activating transmitters.  both  H. EL-BEHEIRY The above of  the  observations  anaesthetic  are c o n s i s t e n t  state  as  a  with the  cumulative  recent  continuum  152  conceptualization  i.e.,  a  series  of  t r a n s f o r m a t i o n s p a s s i n g i n t o each o t h e r , s t a r t i n g w i t h a n a l g e s i a and  emerging  i n t o amnesia and l o s s of n e u r o l o g i c a l  In  responsiveness  t o e x p l o r e t h e v a l i d i t y of t h e h y p o t h e s i s , by an i n v i t r o neuronal  model of  tics  therein.  was  investigated  b l o c k e d the e x c i t a t i o n s u s t a i n e d current intensity brief  (cf.  Fig.  simulated  and the e f f e c t s o f the  anaesthe-  by a c o n t i n u o u s  evoked  at  order  s t a t e was  isoflurane  37 and 3 8 ) .  Glu-depolarizations  the conscious  neocortex Both  (White 1987).  During  and  Althesin  completely  ACh a p p l i c a t i o n w i t h a low this  consistent  time, the  intervals  superimposed  were  slightly  depressed. Because  these  structurally  identical  effects  in t h i s  relevant  to  mechanism  anaesthesia, sustained  the  there  are  ascending  dissimilar  neuronal of  produced  nearly  s u g g e s t i o n s may  state.  specific  i n p u t i n neurons  m o d u l a t i o n may be l a r g e l y  t o modulate t h e r e s p o n s i v e n e s s  following  anaesthetic  pharmacologically  which r e s e t s t h e i r r e s p o n s i v i t i e s This  model, the  the  cholinergic  anaesthetics  That  is,  be  during  alterations  of  w i t h i n the c e r e b r a l  the  cortex  to the s y n a p t i c a l l y released t r a n s m i t t e r s . contingent  on the a b i l i t i e s o f  of t h e p o s t s y n a p t i c  receptors  anaesthetics  of  neocortical  neurons. 9.4  I o n i c mechanisms The e f f e c t s of  of a n a e s t h e t i c  isoflurane  action  and A l t h e s i n  on n e o c o r t i c a l  i n v e s t i g a t i o n s can be summarized a c c o r d i n g t o f i v e b a s i c (1) d e p r e s s i o n of spontaneous  a c t i v i t i e s o f the  neurons  suppression  of  the  "chemical  neurons,  responsiveness"  a p p l i c a t i o n of ACh, G l u , NMDA and GABA near t h e  these  observations:  (2) a t t e n u a t i o n o f evoked e x c i t a t o r y and i n h i b i t o r y s y n a p t i c (3)  in  to  somata,  the  transients, extracellular  H. EL-BEHEIRY (4)  attenuation  K-conductance  of  the  "pharmacologically  ( i . e . AHP g e n e s i s )  thetic  agents  i n c r e a s e s i n t h e [Ca  explanation  interfered  with  receptor-operated Ca-channels  Ca-activated  and  (5) blockade of Glu and K-evoked The most p a r s i m o n i o u s  isolated"  f o r these  Ca-influx  ]..  effects  through  is  t h a t the  a t p r e - and p o s t s y n a p t i c  sites.  the Ca-dependent  would be a t t e n u a t e d ( c f .  Inoui  et al  e t a l . 1987;  G a h w i l e r and Brown  e t a l . 1988;  Bormann 1988,  the  of  and  1983;  Oyama  Gunderson  et  Ca-spikes  1987;  Murphy  A k a i k e 1990).  al  significantly suggested 1974b;  1986),  1984).  This  Krnjevic 1988)  reduced  that  (Heyer  responsible  an  by  the  1978a;  hypoxia  (cf.  that  a  significant  applications 1989).  et a l .  (cf.  increase  of a n a e s t h e t i c s  Section in  1962;  1981)  (Daniell  ].  and  is  was  and H a r r i s  with  (cf.  1988;  it  ].  1988;  be  (Krnjevic*  E c k e r t and  mimick  Chad  simulate  the  the  1971;  effects  and K r n j e v i c  of  1989;  to reconcile with  with  not  are  may  Godfraind et a l .  as  the  (Nishi  Macdonald  [Ca  difficult well  of  Ca-conductances  in  Lablond  6) as 2+  [Ca  suppression  that anaesthetics  1952,  Benavides  Ca-currents  and  Ca-channels  Such an assumption  investigations  1988;  Furthermore,  increase  F u j i w a r a e t a l . 1987;  K r n j e v i c ' and Walz 1 9 9 0 ) . present  (Quastel  Scholfield  together  and  Gross  agents.  i n a c t i v a t i o n of  Vanderkooi  1982)  5)  voltage-dependent  would be e x p e c t e d on the b a s i s  et al  reversible  that  anaesthetic-induced  may l e a d t o  Section  1988;  anaesthetic  a c t i o n s of metabolic i n h i b i t o r s Krnjevic*  Puil  Stafstrom  e t a l . 1986;  The dose-dependent (cf.  for  G l u , NMDA and GABA  and M i l l e r  and Macdonald  and  suggest  t o ACh,  1986; M i s g e l d  i s o l a t e d C a - a c t i v a t e d K-conductance  attenuation  the  responses  and  Consequently,  t h e r e p e t i t i v e s p i k i n g a c t i v i t i e s of t h e neurons a r e d e c r e a s e d ( c f . whereas  anaes-  voltage-dependent  t r a n s m i t t e r r e l e a s e and s l o w inward p o s t s y n a p t i c C a - c u r r e n t s  e t a l . 1985)  153  recent  reports  observed  during  Puil  and  Baimbridge  H. EL-BEHEIRY  Another protein  possibility  involved  Firestone  is  in  1988).  One  that  anaesthetic  secondary  messenger  such c a n d i d a t e  Ca-activated phospholipid-dependent c e l l u l a r proteins enzyme that  has  enzyme  of  et a l .  1988;  the  is  mostly  Sladeczek  (Castagna  et a l .  Firestone  1982;  with  et a l .  1988),  PKC  is  of  of  this  muscarinic  hydrolysis  In  and  (Berridge  increases  the  the b r a i n  this  et a l .  1982)  and i o n channel Moreover,  a  demonstrations  (Kikkawa  can  (cf.  a variety  greatly  synapses  (1988) demonstrated t h a t a n a e s t h e t i c s  i s o l a t e d r a t b r a i n homogenates.  (PKC).  activation.  1989).  cytosolic  The a c t i v i t y of  products  (DeRiemer e t a l . 1985)  Conn  C  phospholipid  its  a  processes  t r a n s d u c t i o n by t h e  et a l .  associated  affects receptor s e n s i t i v i t y  signaling.  initial  o f PKC f o r Ca, t h e r e b y c a u s i n g which  to  transduction  protein kinase  receptor-activated inositol  Peralta  affinity  one  bind  enzyme t h a t p h o s p h o r y l a t e s  been f i r m l y l i n k e d t o s i g n a l  glutamatergic 1987;  is  i n v o l v e d i n transmembrane  diacylglycerol,  molecules  154  activity  Firestone  and  inhibit PKC-activity  A s i m p l i f i e d r e p r e s e n t a t i o n of t h e  in  sequence  of events may be s c h e m a t i z e d as below: Anaesthetics  bind  Voltage-dependent hence NMDA  thereby kinase  C  protein  to  that  the  triggers  C  are  stimulate  no  the  longer  activity  the  already  the would  phosphate  positive  C a - i n f l u x would be b l o c k e d .  protein  (PI)  which  et a l .  activates  channels.  be  the  by  diacylglycerol mechanism  Muscarinic (Malenka  for  receptors  second a  f o r ACh  e t a l . 1986;  cycle protein the  binding.  messenger  further  and  However,  anaesthetic  C. and  1988)  hydrolysis  receptor-operated inhibited  kinase  phosphorylated  Sladeczek  phosphoinositide  feedback  known t o a c t i v a t e P i - h y d r o l y s i s  of  efficiently  i n t r a c e l l u l a r Ca-concentration  inositol a  inhibit  (Sugiyama e t a l . 1987;  phosphorylate  kinase  Therefore  Glu  1987)  increasing  and  Ca-channels  inactivated. (Berridge  with  pathway  increase  in  in the b r a i n  are  P e r a l t a et a l .  1988;  Dutar and N i c o l 1 a  protein  1988)  kinase  in  El-Fakahany  et a l .  between  ionic  the  secondary  during  agents  neocortical  1989). basis  messenger  anaesthetic  and t h e i r e f f e c t s are t h o u g h t  identified 1988). for  on  the  In  lack  of  muscarinic the  of  155  t o be m e d i a t e d  through  and  Gruen  induced  of  inhibition  of  o f ACh-evoked  a p r o t e i n kinase  enzyme.  Indeed the  et a l . induced the  1987;  Sigel  and  i n h i b i t i o n of  GABA-induced  GABA  GABA^ Baur  PKC  by  receptors  1988;  are  phosphoryla-  receptor  has  Gyenes  PKC may be p a r t l y  depolarizations  the  depolarizations  on  the  link  and  the  effects  of  cf.  the  responses  their  region  1988;  understanding  ACh  presumed  addition,  cytoplasmic  (Scholfield  depression  a  (Woody  can e x p l a i n the d e p r e s s i o n  An a n a e s t h e t i c  the  the  systems,  anaesthesia.  site  Despite of  p r o b a b l y mediated t h r o u g h tion  neurons  H. EL-BEHEIRY  been et a l .  responsible  observed  in  these  investigations. 9.5  M o l e c u l a r mechanisms o f a n a e s t h e s i a Because  structures  of of  the  anaesthetic  of d r u g - r e c e p t o r or  protein  models,  Firestone  and  investigations actions of  of  agents  reconciling  dissimilarities  with conventional  anaesthesia.  enjoyed most Kitz  of  1988;  challenge  of a n a e s t h e t i c s  membrane  in  interactions, anaesthesiologists  models  have  difficulty  - - the l i p i d / p r o t e i n controversy  the e x p e r i m e n t a l  Firestone the  are  properties.  c h e m i c a l l y d i s t i n c t agents  Instead,  1988).  assumptions  induced by Moreover, (isoflurane  chemical  key'  concepts  not f a v o u r e d  specific  'lock  have  and  non-specific attention To  some  suggesting  non-specific the  in  similar  t i c s c o u l d a c t s p e c i f i c a l l y a t s i m i l a r hydrophobic  lipid-based  (cf.  Janoff  1982;  extent,  the  present  that  the  biophysical results  and A l t h e s i n )  or  important alterations  obtained  indicate that  sites  anaesthe-  r a t h e r than  t o d i f f e r e n t p r o t e i n p o c k e t s as suggested e a r l i e r ( c f . R i c h a r d s  using  1975;  binding 1983).  H. EL-BEHEIRY  10  1.  The  neurons  of  recording  of  general  layers  IV  and  ness  The  transients  to  anaesthetics  V  of  anaesthetic  techniques,  synaptic  SUMMARY AND CONCLUSIONS  effects  preparations.  156  on  guinea actions  passive  evoked  neocortex  in  were  examined,  using  and  applied  investigated  pig  active  by o r t h o d r o m i c  iontophoretically  were  in  membrane  stimulation,  transmitter  on  pyramidal  vitro  slice  intracellular  properties,  neuronal  substances  the  responsive-  and  postspike  afterhyperpolarizing potentials. 2 . Two s t r u c t u r a l l y d i s s i m i l a r a n a e s t h e t i c s , e t h e r and A l t h e s i n  - a steroidal  passive  membrane  highest  concentrations  hyperpolarized by  10-30%.  produced  However,  The  intracellular  of  used  a  halogenated  p r e p a r a t i o n , d i d not p r o f o u n d l y  a f f e c t the  the  clinically  reversible  repetitive  >2  except MAC  relevant  evoked  by  injections  also  -  high  doses.  Althesin  their  induction  i n the  at  and  mV and i n c r e a s e d  decrease  firing  current-pulse  neurons,  (isoflurane  the neurons by 3-8  a sharp,  neurons.  properties  isoflurane  input  doses  of  The  >300  uM)  conductance both  agents  spontaneous a c t i v i t i e s o f orthodromic were  stimulation  strongly  the  or  by  inhibited  by  anaesthetic a p p l i c a t i o n . 3.  A  observed ~50  dose-dependent, on a p p l i c a t i o n of  uM r e s p e c t i v e l y .  EPSPs  by  the  In  (bicuculline)  this  blockade,  IPSP  in  isoflurane  order  conductances  antagonist  effective  reversible  neocortical with E C s 5 Q  eliminate a possible  involved  anaesthetics  the  of  and A l t h e s i n  in  the  was a p p l i e d t o g e t h e r  the  reducing  to  depression  still  epileptiform  of  shunting  concomitant  EPSPs  was  1 MAC  and  effect  IPSPs,  a  GABA A  with the anaesthetics. depressed  activities  the  evoked  EPSPs by  on  and  After were  bicuculline  H. EL-BEHEIRY  during  subpial  stimulation.  K-conductances anaesthetics IPSPs.  by  internal  IPSPs a l s o  Cs-application  were evoked d u r i n g in  order  that  c o u l d be s t u d i e d more a c c u r a t e l y on t h e  Application  dose-dependent  of  isoflurane  or  Althesin  of  effects  of  the  Cl-mediated  depressed  applications  of  acetylcholine,  these  glutamate,  GABAergic  IPSPs  in  a  and  GABA  Somatic a p p l i c a t i o n of GABA i n d u c e d  only  d e p o l a r i z e d the pyramidal  neurons.  h y p e r p o l a r i z i n g responses  t h a t were e i t h e r d e s e n s i t i z i n g  and were l o n g - l a s t i n g  the d u r a t i o n  responses  blockade  manner.  Dendritic  4.  The  1 5 7  in  layers  if IV  and  V neurons  or  NMDA  nondesensitizing  of GABA a p p l i c a t i o n was exhibited  tachyphylaxis  s.  >10 on  The  frequent  a p p l i c a t i o n of NMDA o r GABA . Isoflurane,  5. the  A l t h e s i n and i n  depolarizing  responses  and  some c a s e s , associated  halothane,  membrane  markedly  reduced  conductance  changes  evoked by d e n d r i t i c a p p l i c a t i o n of a c e t y l c h o l i n e , g l u t a m a t e , and NMDA. hyperpolarization  evoked  by  GABA was  depolarization  was  a c e t y l c h o l ine>  glutamate  and  assessed  the  for  from  just-maximal 1.9  depressed  EC^Q  responses  pronounced  a f f e c t e d whereas  slightly. NMDA>» the  The  GABA.  order This  In  MAC compared  actions  during  depression  with  hypomagnesaemia,  a  clinical  were  determined  on  the  neuronal  responses  to  was  of  the  EC^Q  of  were 7 5 nM  that  of is  anaesthetic known  a s s o c i a t e d w i t h increased a n a e s t h e t i c requirements, the a c t i o n s of tics  also  respectively.  mechanisms  condition  the  was  The s e l e c t i v i t y was  i n the case of A l t h e s i n a c t i o n s where the E C ^ s  pathophysiological  evoked  depression  of t h e g l u t a m a t e - r e s p o n s e s .  order to i n v e s t i g a t e  GABA  selectivity  and 9 0 uM f o r t h e d e p r e s s i o n of both t r a n s m i t t e r responses 6.  the  of  isoflurane-induced  t o ACh w h i c h was 0 . 9  MAC f o r t h e s u p p r e s s i o n  less  not  The  putative  to  be  anaesthe-  transmitters  H. EL-BEHEIRY  during  simulated  acetylcholine decremental  hypomagnesia  or  GABA  manner.  in  were  vitro.  blocked  Hypomagnesia  7. A s t r i k i n g sion  by  same e f f e c t was which  of  K-conductance) GABAergic 8.  evoked  in  isoflurane  GABA a c t i o n s  afterhyperpolarization  conditions  (produced  the  Fura-2  of  IPSP-blockade  through  activation  a and  were  more  hyperpolarizing,  Cl-dependent  a  suppres-  (AHP).  by  of  The  bicuculline, Ca- a c t i v a t e d  influence  m i c r o s p e c t r o f l u o r i m e t r y , the a c t i o n s  on  g l u t a m a t e - and K-evoked 2+ i n c r e a s e s i n [Ca J . under  quisqualate- receptors  would  The v o l a t i l e a n a e s t h e t i c 2+ [Ca  i.e.,  of  ]  by  due  to  activity.  Using  studied  effects  [Mg  study was t h e dose-dependent  postspike  under  AHPs  from  responses,  in this  the  observed  a l l e v i a t e d the  the  external  evoked  glutamate.  observation  anaesthetics  depolarizations  lowering  reversed  A l t h e s i n on t h e GABA and g l u t a m a t e depressed than those o f  by  The  158  ].  induced  by  be  also  increases conditions  favoured,  depressed  extracellular  in  K  2+  where  were  the  [Ca  of  isoflurane  ]..  The  actions  attenuated  under  NMDA- o r  isoflurane.  voltage-dependent  applications  glutamate  at  by  were  increases  conditions  in  where  N a - s p i k e g e n e s i s was b l o c k e d by TTX. 9. These  investigations  excitability attenuation suggested  in of  that  interfering mechanisms  neocortex  induced  excitation these  rather  agents  specifically of  p r o v i d e e v i d e n c e t h a t the d e p r e s s i o n  behavioural  synaptic  Ca-conductance  together  with  by than  decrease  with  the  attenuations  of  is  and  the  to  lt  is  cortical  activity  by  transmitters  involved  in  level of  crucial the  to  EPSPs,  of  of  The d e p r e s s i o n the IPSPs  Althesin  neuronal due  potentiation  actions  arousal. which  isoflurane  of  of  is  inhibition.  the  "isolated"  generation and  the  of  post-  the  glutamate  AHPs or  H. EL-BEHEIRY K-induced the  increases  processes  interference Ca-channels.  in  generating  [Ca  2+  ]..,  suggest  consciousness  with Ca-influx  through  that in  the  the  anaesthetic  neocortex  voltage-dependent  and  may  actions result  159 on from  receptor-operated  H. EL-BEHEIRY 11  160  REFERENCES  Adams, PR. 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